US10767175B2 - High specificity genome editing using chemically modified guide RNAs - Google Patents

High specificity genome editing using chemically modified guide RNAs Download PDF

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US10767175B2
US10767175B2 US15/493,129 US201715493129A US10767175B2 US 10767175 B2 US10767175 B2 US 10767175B2 US 201715493129 A US201715493129 A US 201715493129A US 10767175 B2 US10767175 B2 US 10767175B2
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sequence
modification
guide
nucleotide
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US20170355985A1 (en
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Douglas J Dellinger
Daniel E Ryan
Subhadeep Roy
Jeffrey R Sampson
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Agilent Technologies Inc
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Agilent Technologies Inc
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Priority to AU2017277918A priority patent/AU2017277918B2/en
Priority to CN201780049055.3A priority patent/CN109563514B/zh
Priority to PCT/US2017/036648 priority patent/WO2017214460A1/en
Priority to KR1020197000571A priority patent/KR102382772B1/ko
Priority to EP17811048.2A priority patent/EP3469084A4/en
Priority to CN202310155019.2A priority patent/CN116676305A/zh
Priority to CA3026372A priority patent/CA3026372A1/en
Priority to JP2018564259A priority patent/JP7093728B2/ja
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Definitions

  • the present invention relates to the field of molecular biology.
  • the present invention relates to the clusters of regularly interspaced short palindromic repeats (CRISPR) technology.
  • CRISPR regularly interspaced short palindromic repeats
  • the native prokaryotic CRISPR-Cas system comprises an array of short repeats with intervening variable sequences of constant length (i.e., clusters of regularly interspaced short palindromic repeats, or “CRISPR”), and CRISPR-associated (“Cas”) proteins.
  • CRISPR regularly interspaced short palindromic repeats
  • Cas CRISPR-associated proteins.
  • the RNA of the transcribed CRISPR array is processed by a subset of the Cas proteins into small guide RNAs, which generally have two components as discussed below. There are at least six different systems: Type I, Type II, Type III, Type IV, Type V and Type VI.
  • the enzymes involved in the processing of the RNA into mature crRNA are different in the six systems.
  • the guide RNA comprises two short, non-coding RNA species referred to as CRISPR RNA (“crRNA”) and trans-acting RNA (“tracrRNA”).
  • the gRNA forms a complex with a Cas nuclease.
  • the gRNA:Cas nuclease complex binds a target polynucleotide sequence having a protospacer adjacent motif (“PAM”) and a protospacer, which is a sequence complementary to a portion of the gRNA.
  • PAM protospacer adjacent motif
  • the recognition and binding of the target polynucleotide by the gRNA:Cas nuclease complex induces cleavage of the target polynucleotide.
  • the native CRISPR-Cas system functions as an immune system in prokaryotes, where gRNA:Cas nuclease complexes recognize and silence exogenous genetic elements in a manner analogous to RNAi in eukaryotic organisms, thereby conferring resistance to exogenous genetic elements such as plasmids and phages.
  • sgRNA single-guide RNA
  • gRNA including a sgRNA
  • Specificity refers to the ability of a particular gRNA:Cas nuclease complex to bind, nick, and/or cleave a desired target sequence, whereas less or no binding, nicking, and/or cleavage of polynucleotides different in sequence and/or location from the desired target occurs.
  • specificity refers to minimizing off-target effects of the gRNA:Cas nuclease complex.
  • gRNA including sgRNA, having desired binding affinity for the target polynucleotide with reduced off-target effects while, nonetheless, having desired gRNA functionality.
  • FIG. 1 is an illustration of an exemplary CRISPR-Cas system.
  • a complex is formed by a single-guide RNA and a Cas protein, and the complex recognizes and binds a target polynucleotide.
  • the Cas nuclease is the S. pyrogenes Cas9 nuclease.
  • the S. pyrogenes Cas9 nuclease recognizes a PAM sequence (here, the PAM sequence is a 3-nucleotide sequence of NGG, where N is A, G, C or T, but other PAM sequences are known to exist, such as NAG and others).
  • the sgRNA includes a guide sequence, a crRNA sequence or segment, and tracrRNA sequence or segment. The guide sequence of the sgRNA hybridizes with the DNA target directly upstream of the PAM sequence.
  • FIG. 3 shows the structures of some of the various chemical modifications that can be included in a guide RNA; of course, FIG. 3 is not intended to be limiting and many other modifications as described herein can be employed.
  • FIG. 5A illustrates a single-guide RNA (sgRNA) or a two-piece dual-guide RNA (“dgRNA”) wherein a crRNA segment and a tracrRNA segment form a hybridized duplex and shows the guide RNA's extension region, locking region, sampling region, seed region (typically a 10-mer), a dual-guide stem or a single-guide stem-loop, and a tracrRNA region.
  • FIG. 5B illustrates how, after the initial formation of a seed duplex between the seed portion of a guide sequence and a complementary DNA sequence, the binding of the nucleotides proceeds sequentially through the sampling region and locking region to form a RNA/DNA duplex having a melting temperature.
  • FIG. 5A illustrates a single-guide RNA (sgRNA) or a two-piece dual-guide RNA (“dgRNA”) wherein a crRNA segment and a tracrRNA segment form a hybridized duplex and shows the guide RNA's extension region, locking region, sampling region
  • FIG. 5C illustrates that chemical modifications in nucleotides of the seed region can decrease the binding energy of individual base pairs while retaining a high level of cooperativity, thereby extending the sampling region and lowering the melting temperature of the RNA/DNA duplex.
  • FIG. 5C also illustrates chemical modification in the sampling region.
  • FIG. 6A illustrates experimental crRNA polynucleotides with 20-nucleotide guide sequences and modified by various types of chemical modifications incorporated at various positions in the guide sequence.
  • FIG. 6B shows the melting curve for an RNA/DNA duplex comprising an unmodified crRNA.
  • FIGS. 6C through 6F show melting curves for RNA/DNA duplexes comprising chemically modified crRNAs comprising different types of modifications at nucleotides 6 through 9 in distinct guide sequences.
  • FIG. 7 is a graph showing change in melting temperature of a 20-base pair duplex of guide sequence (in a crRNA) hybridized to a complementary DNA strand after incremental 5′ truncation or 5′ extension of the guide sequence as indicated on the x-axis by negative or positive integers, respectively.
  • FIG. 8A shows the impact of chemical modifications in gRNAs targeted to the “CLTA1” sequence in the human CLTA gene with regard to in vitro cleavage of target polynucleotide called “ON” and separately assayed cleavage of two different off-target polynucleotides called “OFF1” and “OFF3,” representing CLTA1 ON-target, CLTA1 OFF1-target, and CLTA1 OFF3-target, respectively.
  • FIG. 8B is derived from the cleavage results in FIG. 8A by calculating a ratio of cleaved target polynucleotide versus cleaved off-target polynucleotide for each synthetic sgRNA assayed. Also calculated is a “Specificity Score” obtained by multiplying each ratio by the respective ON-target cleavage percentage per guide RNA assayed. The shaded ratios and Specificity Scores are notable for their larger values with respect to the others shown in FIG. 8B .
  • FIG. 9A shows the impact of 2′-O-methyl-3′-PACE (“MP”) modifications at various locations in gRNAs targeted to the “CLTA4” sequence in the human CLTA gene with regard to in vitro cleavage of target polynucleotide called “ON” and separately assayed cleavage of three different off-target polynucleotides called “OFF1”, “OFF2”, and “OFF3,” representing CLTA4 ON-target, CLTA4 OFF1-target, CLTA4 OFF2-target, and CLTA4 OFF3-target, respectively.
  • MP 2′-O-methyl-3′-PACE
  • FIG. 9B is derived from the cleavage results in FIG. 9A by calculating a ratio of cleaved target polynucleotide versus cleaved off-target polynucleotide for each synthetic sgRNA assayed. Specificity Scores are calculated as described for FIG. 8B , and the scores greater than or equal to 1.5 are shaded. The three highest scores per off-target polynucleotide are indicated by darker shading.
  • FIG. 10 shows the impact of MP modifications at various locations in gRNAs targeted to a sequence in the human IL2RG gene with regard to in vitro cleavage of target polynucleotide called “ON” and separately assayed cleavage of an off-target polynucleotide called “OFF3” in this figure representing IL2RG ON-target and IL2RG OFF3-target, respectively.
  • a ratio is calculated for cleaved target polynucleotide versus cleaved off-target polynucleotide for each synthetic sgRNA assayed.
  • Specificity Scores are calculated as described for FIG. 8B , and the scores greater than 2.0 are shaded. The three highest scores are indicated by darker shading.
  • FIG. 11A shows the impact of MP modifications at various locations in gRNAs targeted to a sequence in the human HBB gene with regard to in vitro cleavage of target polynucleotide called “ON” and separately assayed cleavage of an off-target polynucleotide called “OFF1” in this figure representing HBB ON-target and HBB OFF1-target, respectively.
  • a ratio is calculated for cleaved target polynucleotide versus cleaved off-target polynucleotide for each synthetic sgRNA assayed.
  • Specificity Scores are calculated as described for FIG. 8B , and the scores greater than 2.0 are shaded. The three highest scores are indicated by darker shading.
  • FIG. 11B shows the impact of various types of modifications in gRNAs targeted to a sequence in the human HBB gene in human K562 cells transfected with synthetic sgRNA and Cas9-expressing plasmid with regard to cleavage of a target genomic locus called “ON” versus concurrent cleavage of three different off-target genomic loci called “OFF1”, “OFF2” and “OFF3” in this figure representing endogenous HBB ON-target, HBB OFF1-target, HBB OFF2-target and HBB OFF3-target sites, respectively.
  • “Unmodif” indicates an sgRNA that was not chemically modified.
  • 3xM indicates that 2′-O-methyl (“M”) nucleotide was incorporated at the very first three and the very last three nucleotides of an sgRNA strand, namely at its 5′ and 3′ termini respectively.
  • 3 ⁇ MS indicates that 2′-O-methyl-3′-phosphorothioate (“MS”) nucleotide was incorporated likewise at the 5′ and 3′ termini of an sgRNA
  • 3 ⁇ MSP indicates that 2′-O-methyl-3′-thioPACE (“MSP”) nucleotide was incorporated likewise at the 5′ and 3′ termini of an sgRNA. All sgRNAs were assayed for editing of the indicated loci in transfected cells.
  • FIG. 12A shows the same results in entries 1-17 as shown in FIG. 11A , with the entries ranked according to Specificity Score from highest to lowest.
  • Entries 18-64 show the impact of additional MP modifications at various locations in gRNAs targeted to a sequence in the human HBB gene with regard to in vitro cleavage of target polynucleotide called “ON” and separately assayed cleavage of an off-target polynucleotide called “OFF1,” representing HBB ON-target and HBB OFF1-target, respectively.
  • “1 ⁇ MP” indicates that the terminal nucleotides at both the 5′ and 3′ ends have been modified with MP.
  • a ratio is calculated for cleaved target polynucleotide versus cleaved off-target polynucleotide for each synthetic sgRNA assayed.
  • Specificity Scores are calculated as described for FIG. 8B . The highest scores are shaded.
  • FIG. 12B shows the impact of MP modifications at various locations in gRNAs targeted to a sequence in the human HBB gene in transfected cells with regard to cleavage of a target genomic locus called “ON” and concurrent cleavage of an off-target genomic locus called “OFF1” in this figure representing endogenous HBB ON-target and HBB OFF1-target sites respectively.
  • the percentage of cleavage yielded at either or both sites in cultured cells transfected with a complex of synthetic sgRNA and recombinant Cas9 protein is determined 48 hours post-transfection by PCR amplification of the on-target and off-target loci in split samples of purified genomic DNA, followed by next-generation sequencing of pooled amplicons and bioinformatic partitioning of the sequence reads according to the presence versus absence of an indel near the on-target or off-target cleavage site being evaluated. Indels generated in each sample of transfected cells are normalized relative to a control sample of mock-transfected cells treated with buffer instead of sgRNA:Cas9 complex.
  • a ratio is calculated for the number of sequence reads showing a target site indel versus the number of reads showing an off-target site indel for each sgRNA transfected separately.
  • Specificity Scores are calculated as described for FIG. 8B . “1 ⁇ MP” indicates that the terminal nucleotides at both the 5′ and 3′ ends have been modified with MP.
  • Entries 1-21 show results obtained by transfecting and culturing K562 cells, whereas entries 22-42 show results obtained by transfecting and culturing induced pluripotent stem cells (also known as iPS cells or iPSCs).
  • Entries 1-12 are ranked according to Specificity Score from highest to lowest. Likewise entries 13-19, entries 22-33, and entries 34-40 are ranked by Specificity Score per grouping. Specificity Scores greater than 2.0 are shaded.
  • FIG. 12C shows an alternative organization of the results in FIG. 12B according to the measured ratios, sorted from highest to lowest ratio per grouping.
  • FIG. 13 shows a comparison of the results presented in FIGS. 9A, 9B, 10 and 11A .
  • FIG. 13 shows several trends from studies of chemically modified guide RNAs with respect to target specificity when used in a Cas system for cleaving target polynucleotides. The concepts supported by the trends are especially useful when off-target polynucleotides are also present in the Cas system.
  • FIG. 14 shows the impact of various types of modifications in IL2RG sgRNAs and VEGFA sgRNAs in K562 cells.
  • This invention is based, at least in part, on an unexpected discovery that certain chemical modifications to gRNAs are tolerated by the CRISPR-Cas system and decrease the off-target effects of Cas:gRNA complexation without substantially compromising the efficacy of Cas:gRNA binding to, nicking of, and/or cleavage of the target polynucleotide.
  • This invention provides synthetic guide RNAs comprising at least one specificity-enhancing modification.
  • the at least one specificity-enhancing modification weakens or strengthens the association of at least one nucleotide pair between the synthetic guide RNA and the target polynucleotide and/or weakens the association of at least one nucleotide pair between the synthetic guide RNA and at least one off-target polynucleotide such that at least one of the off-target weakenings is greater than the on-target weakening if present.
  • the synthetic guide RNA has gRNA functionality.
  • the specificity-enhancing modification(s) can be located in the guide sequence, for example, in the locking region, sampling region, and/or seed region.
  • the specificity-enhancing modification(s) lowers melting temperatures of duplexes formed by the gRNA and a target polynucleotide sequence and off-target polynucleotide sequence(s), or raises melting temperature of the gRNA/target duplex and lowers melting temperature of at least one gRNA/off-target duplex.
  • This invention also provides gRNA:Cas protein complex comprising these synthetic guide RNAs, methods for cleaving, nicking or binding target polynucleotides using the synthetic guide RNAs, and sets, libraries, kits and arrays comprising the synthetic guide RNAs. This invention also provides method of preparing synthetic guide RNAs.
  • guide RNA generally refers to an RNA molecule (or a group of RNA molecules collectively) that can bind to a Cas protein and aid in targeting the Cas protein to a specific location within a target polynucleotide (e.g., a DNA or an mRNA molecule).
  • a guide RNA can comprise a crRNA segment and a tracrRNA segment.
  • crRNA refers to an RNA molecule or portion thereof that includes a polynucleotide-targeting guide sequence, a stem sequence (for additional clarity: the stem sequence encompasses a stem-forming sequence which, in a single guide RNA, forms a stem with a corresponding part of tracrRNA), and, optionally, a 5′-overhang sequence.
  • tracrRNA refers to an RNA molecule or portion thereof that includes a protein-binding segment (e.g., the protein-binding segment is capable of interacting with a CRISPR-associated protein, such as a Cas9).
  • the tracrRNA also includes a segment that hybridizes partially or completely to the crRNA.
  • guide RNA encompasses a single-guide RNA (sgRNA), where the crRNA segment and the tracrRNA segment are located in the same RNA molecule or strand.
  • guide RNA also encompasses, collectively, a group of two or more RNA molecules, where the crRNA segment and the tracrRNA segment are located in separate RNA molecules.
  • guide RNA also encompasses an RNA molecule or suitable group of molecular segments that binds a Cas protein other than Cas9 (e.g., Cpf1 protein) and that possesses a guide sequence within the single or segmented strand of RNA comprising the functions of a guide RNA which include Cas protein binding to form a gRNA:Cas protein complex capable of binding, nicking and/or cleaving a complementary sequence (or “target sequence”) in a target polynucleotide.
  • a guide RNA which include Cas protein binding to form a gRNA:Cas protein complex capable of binding, nicking and/or cleaving a complementary sequence (or “target sequence”) in a target polynucleotide.
  • guide sequence refers to a contiguous sequence of nucleotides in a guide RNA which has partial or complete complementarity to a target sequence in a target polynucleotide and can hybridize to the target sequence by base pairing facilitated by a Cas protein.
  • a target sequence is adjacent to a PAM site (the PAM sequence, and its complementary sequence on the other strand, together constitute a PAM site).
  • NGS Immediately upstream of the PAM sequence
  • the target sequence, which hybridizes to the guide sequence is immediately downstream from the complement (CCN for cas9) of the PAM sequence.
  • Nucleotide 1 of the guide sequence is complementary to the last nucleotide of the target sequence, while the last nucleotide of the guide sequence (nucleotide 20 of the guide sequence in FIG. 1 ) is complementary to the first nucleotide of the target sequence, which is next to the PAM site (and immediately downstream from the complement of the PAM sequence).
  • the location of the target sequence, which hybridizes to the guide sequence may be upstream from the complement of the PAM sequence.
  • a guide sequence can be as short as about 10 nucleotides and as long as about 30 nucleotides. Typical guide sequences are 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24 nucleotides long. Synthetic guide sequences are usually 20 nucleotides long, but can be longer or shorter. When a guide sequence is shorter than 20 nucleotides, it is typically a deletion from the 5′-end compared to a 20-nucleotide guide sequence.
  • a guide sequence may consist of 20 nucleotides complementary to a target sequence. In other words, the guide sequence is identical to the 20 nucleotides upstream of the PAM sequence, except the A/U difference between DNA and RNA.
  • nucleotide 4 of the 20-nucleotide guide sequence now becomes nucleotide 1 in the 17-mer
  • nucleotide 5 of the 20-nucleotide guide sequence now becomes nucleotide 2 in the 17-mer
  • the new position is the original position minus 3.
  • a guide sequence may hybridize to more than 20 nucleotides at the target site, and the additional nucleotides are located at the 5′-end of the guide sequence, because the 3′-end of the guide sequence is complementary to the target next to the PAM site.
  • a guide sequence consists of nucleotides 1 through “20 minus N” (20-N) counting from the 5′-end, wherein N is a positive or negative integer between ⁇ 10 and 10 (optionally between ⁇ 10 and 6).
  • N is a positive or negative integer between ⁇ 10 and 10 (optionally between ⁇ 10 and 6).
  • a given nucleotide position within the guide sequence will be noted as “Position Number Minus N” (number-N).
  • nucleotide at position 5 will be noted as “5-N” (5 minus N), to indicate the shift of position 5 from the reference position obtained from a 20 nucleotides guide sequence that occurs when the guide sequence is truncated or extended by N nucleotides at the 5′-end. Nucleotide positions are positive integers. Thus, any (Number-N) position that is negative or zero is moot, and should be ignored.
  • a guide sequence can be positioned anywhere within the strand or strands that constitute a gRNA. Typical guide sequences are located at or near the 5′ end or the 3′ end of a gRNA strand.
  • Scaffold refers to the portions of guide RNA molecules comprising sequences which are substantially identical or are highly conserved across natural biological species. Scaffolds include the tracrRNA segment and the portion of the crRNA segment other than the polynucleotide-targeting guide sequence (repeat portion) at or near the 3′ end of the crRNA segment.
  • nucleic acid refers to a DNA molecule, an RNA molecule, or analogs thereof.
  • nucleic acid include, but are not limited to DNA molecules such as cDNA, genomic DNA, plasmid or vector DNA or synthetic DNA and RNA molecules such as a guide RNA, messenger RNA or synthetic RNA.
  • the terms “nucleic acid” and “polynucleotide” include single-stranded and double-stranded forms.
  • oligonucleotides, polynucleotides, RNA molecules, distinct strands of DNA molecules, and various nucleic acids comprising 2 or more nucleotides are generally numbered from their 5′ ends, and this convention is used throughout, including instances of 5′ extensions or “overhangs” covalently linked to such molecules.
  • modification refers to a chemical moiety, or portion of a chemical structure, which differs from that found in the four most common natural ribonucleotides: adenosine, guanosine, cytidine, and uridine ribonucleotides.
  • modification refers to a molecular change in, or on, the most common natural molecular structure of adenosine, guanosine, cytidine, or uridine ribonucleotide.
  • modification may refer to a change in or on a nucleobase, in or on a sugar, and/or in an internucleotide phosphodiester linkage.
  • modification may refer to a chemical structural change in a ribonucleotide that occurs in nature such as a chemical modification that occurs in natural transfer RNAs (tRNAs), for example but not limited to 2′-O-Methyl, 1-Methyladenosine, 2-Methyladenosine, I-Methylguanosine, 7-Methylguanosine, 2-Thiocytosine, 5-Methylcytosine, 5-Formylcytosine, Pseudouridine, Dihydrouridine, Ribothymidine, or the like.
  • tRNAs natural transfer RNAs
  • modification may refer to a chemical modification that is not typically found in nature, for example but not limited to 2′-Fluoro, 2′-O-Methoxyethyl, 2′-O-Phenyl, or the like.
  • standard modification refers to the same type of chemical modification in or on a sugar, or in an internucleotide linkage; for example, a 2′-O-Methyl may be attached to the 2′ position of an adenosine, guanosine, cytidine, and/or uridine ribonucleotide, and such modifications may be referred to as the same type of modification or the “same modification.”
  • modifications to nucleobases would only be identified as the “same modification” if the modified nucleobases were composed of the same molecular structure.
  • RNA may comprise three modified nucleotides, for example a guanosine and two cytidines, each modified by 2′-O-Methyl-3′-Phosphonoacetate, and such nucleotides may accurately be described as modified in an identical manner or modified with the same modification.
  • RNA may comprise a 5-Methylcytidine as well as a cytidine that lacks a 5-methyl substituent, and both cytidine nucleotides may be modified by 2′-O-Methyl-3′-Phosphonoacetate, nonetheless these two cytidine nucleotides comprise different modifications and would not be referred to as modified in an identical manner.
  • modified nucleotide in the context of an oligonucleotide or polynucleotide includes but is not limited to (a) end modifications, e.g., 5′ end modifications or 3′ end modifications, (b) nucleobase (or “base”) modifications, including replacement or removal of bases, (c) sugar modifications, including modifications at the 2′, 3′, and/or 4′ positions, and (d) backbone modifications, including modification or replacement of the phosphodiester linkages.
  • modified nucleotide generally refers to a nucleotide having a modification to the chemical structure of one or more of the base, the sugar, and the phosphodiester linkage or backbone portions, including nucleotide phosphates. Chemical modifications to guide RNA are disclosed in U.S. patent application Ser. No. 14/757,204, filed Dec. 3, 2015, the entire contents of which are incorporated by reference herein.
  • xA”, “xG”, “xC”, “xT”, “xU”, or “x(A,G,C,T,U)” and “yA”, “yG”, “yC”, “yT”, “yU”, or “y(A,G,C,T,U)” refer to nucleotides, nucleobases, or nucleobase analogs as described by Krueger et al., “Synthesis and Properties of Size-Expanded DNAs: Toward Designed, Functional Genetic Systems”, (2007) Acc. Chem. Res. 40, 141-50, the contents of which is hereby incorporated by reference in its entirety.
  • Unstructured Nucleic Acid refers to nucleotides, nucleobases, or nucleobase analogs as described in U.S. Pat. No. 7,371,580, the contents of which is hereby incorporated by reference in its entirety.
  • An unstructured nucleic acid, or UNA, modification is also referred to as a “pseudo-complementary” nucleotide, nucleobase or nucleobase analog (see e.g., Lahoud et al. (1991) Nucl. Acids Res. 36:10, 3409-19).
  • PACE internucleotide phosphodiester linkage analogs containing phosphonoacetate or thiophosphonoacetate groups, respectively. These modifications belong to a broad class of compounds comprising phosphonocarboxylate moiety, phosphonocarboxylate ester moiety, thiophosphonocarboxylate moiety and thiophosphonocarboxylate ester moiety.
  • linkages can be described respectively by the general formulae P(CR 1 R 2 ) n COOR and (S)—P(CR 1 R 2 ) n COOR wherein n is an integer from 0 to 6 and each of R 1 and R 2 is independently selected from the group consisting of H, an alkyl and substituted alkyl.
  • M is used herein to indicate a 2′-O-methyl modification
  • S is used herein to indicate a 3′-phosphorothioate internucleotide linkage modification
  • P is used herein to indicate a 3′-phosphonoacetate (or PACE) internucleotide linkage modification
  • MS is used herein to indicate a 2′-O-methyl-3′-phosphorothioate internucleotide linkage modification
  • MP is used herein to indicate an 2′-O-methyl-3′-phosphonoacetate (or 2′-O-methyl-3′-PACE) internucleotide linkage modification
  • MSP is used herein to indicate a 2′-O-methyl-3′-thiophosphonoacetate internucleotide linkage modification.
  • “Sugar pucker” refers to a sugar ring that is non-planar due to steric forces causing one or two atoms of a 5-membered sugar ring to be out of plane. In ribofuranose, the plane C1′-O4′-C4′ is fixed. Endo-pucker means that C2′ or C3′ are turned out of this plane into the direction of O5′. Exo-pucker describes a shift in the opposite direction. C2′-endo and C3′-endo are naturally in equilibrium, but chemical modification can drive the sugar to a preferred pucker. In RNA C3′-endo conformation is predominant. DNA may adjust and is able to take on both conformations.
  • seed region refers to the region of the guide sequence that is complementary to a target nucleic acid sequence which initiates hybridization of the guide sequence to the target nucleic acid sequence. In some cases, the seed region forms a quasi-stable duplex that is aided by a protein, peptide, or protein complex.
  • seed region in a guide sequence of a gRNA consists of nucleotides 11 through 20 in a 20-nucleotide guide sequence, counted from the 5′ end of the guide sequence, but the region can run shorter or longer depending on the nucleotide sequence and on chemical modifications on the RNA nucleotides in this region or through modification of the associated peptides, proteins or protein complexes.
  • sampling region refers to the region adjacent to the seed region, and the binding of these nucleotides proceeds until the binding energy of the duplex is equivalent to the temperature at which the binding is occurring.
  • sampling region in a gRNA consists of nucleotides 5 through 10 in a 20-nucleotide guide sequence, counted from the 5′ end of the guide sequence, unless otherwise indicated, as may be the case when one or more modifications functionally extend the sampling region, thereby encompassing nucleotides 1 through 10, alternatively 2 through 10, alternatively 3 through 10, alternatively 4 through 10, alternatively 1 through 11, alternatively 2 through 11, alternatively 3 through 11, alternatively 4 through 11, alternatively 5 through 11, alternatively 1 through 12, alternatively 2 through 12, alternatively 3 through 12, in the guide sequence.
  • locking region refers to the region adjacent to the sampling region in which the binding energy of the duplex formed is above the temperature at which the binding is occurring.
  • locking region in a gRNA consists of nucleotides 1 through 4 in a 20-nucleotide guide sequence, counted from the 5′ end of the guide sequence, unless otherwise indicated, as may be the case when one or more modifications functionally shorten the locking region to nucleotide 1, alternatively nucleotides 1 through 2, alternatively nucleotides 1 through 3, at the 5′ end of a guide sequence.
  • the locking region can extend beyond the 20-nucleotide length of typical guide sequences of CRISPR-Cas9 systems if one or more nucleotides are covalently linked to the 5′ end to extend the guide sequence from the typical 20 nucleotides to 21 nucleotides, alternatively to 22 nucleotides, alternatively to 23 nucleotides, alternatively to 24 nucleotides, alternatively to 25 nucleotides or more.
  • target polynucleotide refers to a polynucleotide containing a target nucleic acid sequence.
  • a target polynucleotide may be single-stranded or double-stranded, and, in certain embodiments, is double-stranded DNA. In certain embodiments, the target polynucleotide is single-stranded RNA.
  • a “target nucleic acid sequence” or “target sequence,” as used herein, means a specific sequence or the complement thereof that one wishes to bind to, nick, or cleave using a CRISPR system.
  • an “off-target polynucleotide” or “off-target” refers to a polynucleotide containing a partially homologous acid sequence to the intended target nucleic acid.
  • An off-target polynucleotide may be single-stranded or double-stranded, and, in certain embodiments, is double-stranded DNA.
  • An “off-target nucleic acid sequence” or “off-target sequence,” as used herein, means a specific sequence or the complement thereof that one does not wish to bind to, nick, or cleave using a CRISPR system and that has substantial sequence identity with, but is not identical to, a target nucleic acid sequence.
  • an off-target nucleic acid sequence has substantial sequence identity with a target nucleic acid sequence when it has at least about 60%, at least about 75%, at least about 85%, at least about 90%, at least about 90-95%, at least about 97%, or more nucleotide (or amino acid) sequence identity.
  • HBB polynucleotide refers to any polynucleotide that comprises at least a portion of the genes HBB, VEGFA, IL2RG, CLTA1 or CLTA4, respectively.
  • polynucleotides include naturally occurring, recombinant, or synthetic polynucleotides.
  • polynucleotides may include polynucleotide sequences found at the locus in the genome associated with such genes, and accordingly encompasses alleles and variants of such genes.
  • the term “specificity” refers to how well a guide RNA is able to distinguish between ON target polynucleotides and one or more OFF target polynucleotides.
  • the specificity of a guide RNA can be determined by, for example, calculating an ON target cleaving, binding, or nicking percentage as well as an OFF target cleaving, binding, or nicking percentage; calculating an ON:OFF ratio; and/or a specificity score derived from comparable ON and OFF target percentages (see Examples of this disclosure).
  • specificity score refers to a number obtained by multiplying an ON:OFF ratio by its respective ON-target percentage per guide RNA assayed; by way of example, a guide RNA having a 80% ON target percentage and a 8% OFF target percentage yields an ON:OFF ratio of 10 and has a specificity score of 8.
  • binding or nicking is assessed using cleaving as a surrogate; for example, in an assay where indel formation at a target site is quantified by sequencing to assess cleavage, such assay can be used to assess binding or nicking activity of the gRNA as well.
  • hybridization or “hybridizing” refers to a process where completely or partially complementary polynucleotide strands come together under suitable hybridization conditions to form a double-stranded structure or region in which the two constituent strands are joined by hydrogen bonds.
  • partial hybridization includes where the double-stranded structure or region contains one or more bulges or mismatches.
  • CRISPR-associated protein refers to a wild type Cas protein, a fragment thereof, or a mutant or variant thereof.
  • the term “Cas mutant” or “Cas variant” refers to a protein or polypeptide derivative of a wild type Cas protein, e.g., a protein having one or more point mutations, insertions, deletions, truncations, a fusion protein, or a combination thereof.
  • the “Cas mutant” or “Cas variant” substantially retains the nuclease activity of the Cas protein.
  • the “Cas mutant” or “Cas variant” is mutated such that one or both nuclease domains are inactive.
  • the “Cas mutant” or “Cas variant” has nuclease activity. In certain embodiments, the “Cas mutant” or “Cas variant” lacks some or all of the nuclease activity of its wild-type counterpart.
  • CRISPR-associated protein or “Cas protein” also includes a wild type Cpf1 protein of various species of prokaryotes (and named for Clustered Regularly Interspaced Short Palindromic Repeats from Prevotella and Francisella 1 ribonucleoproteins or CRISPR/Cpf1 ribonucleoproteins), a fragment thereof, or a mutant or variant thereof.
  • nuclease domain of a Cas protein refers to the polypeptide sequence or domain within the protein which possesses the catalytic activity for DNA cleavage. Cas9 typically catalyzes a double-stranded break upstream of the PAM sequence.
  • a nuclease domain can be contained in a single polypeptide chain, or cleavage activity can result from the association of two (or more) polypeptides.
  • a single nuclease domain may consist of more than one isolated stretch of amino acids within a given polypeptide.
  • Examples of these domains include RuvC-like motifs (amino acids 7-22, 759-766 and 982-989 in SEQ ID NO: 1) and HNH motif (amino acids 837-863); see Gasiunas et al. (2012) Proc. Natl. Acad. Sci. USA 109:39, E2579-E2586 and WO2013176772.
  • a synthetic guide RNA that has “gRNA functionality” is one that has one or more of the functions of naturally occurring guide RNA, such as associating with a Cas protein, or a function performed by the guide RNA in association with a Cas protein.
  • the functionality includes binding a target polynucleotide.
  • the functionality includes targeting a Cas protein or a gRNA:Cas protein complex to a target polynucleotide.
  • the functionality includes nicking a target polynucleotide.
  • the functionality includes cleaving a target polynucleotide.
  • the functionality includes associating with or binding to a Cas protein.
  • the Cas protein may be engineered to be a “dead” Cas protein (dCas) fused to one or more proteins or portions thereof, such as a transcription factor enhancer or repressor a deaminase protein etc., such that the one or more functions is/are performed by the fused protein(s) or portion(s) thereof.
  • the functionality is any other known function of a guide RNA in a CRISPR-Cas system with a Cas protein, including an artificial CRISPR-Cas system with an engineered Cas protein.
  • the functionality is any other function of natural guide RNA.
  • the synthetic guide RNA may have gRNA functionality to a greater or lesser extent than a naturally occurring guide RNA.
  • a synthetic guide RNA may have greater functionality as to one property and lesser functionality as to another property in comparison to a similar naturally occurring guide RNA.
  • a Cas protein having a single-strand “nicking” activity refers to a Cas protein, including a Cas mutant or Cas variant, that has reduced ability to cleave one of two strands of a dsDNA as compared to a wild type Cas protein.
  • a Cas protein having a single-strand nicking activity has a mutation (e.g., amino acid substitution) that reduces the function of the RuvC domain (or the HNH domain) and as a result reduces the ability to cleave one strand of the target DNA.
  • mutations e.g., amino acid substitution
  • examples of such variants include the D10A, H839A/H840A, and/or N863A substitutions in S. pyogenes Cas9, and also include the same or similar substitutions at equivalent sites in Cas9 enzymes of other species.
  • a Cas protein having “binding” activity or that “binds” a target polynucleotide refers to a Cas protein which forms a complex with a guide RNA and, when in such a complex, the guide RNA hybridizes with another polynucleotide, such as a target polynucleotide sequence, via hydrogen bonding between the bases of the guide RNA and the other polynucleotide to form base pairs.
  • the hydrogen bonding may occur by Watson Crick base pairing or in any other sequence specific manner.
  • the hybrid may comprise two strands forming a duplex, three or more strands forming a multi-stranded triplex, or any combination of these.
  • CRISPR function means any function or effect that can be achieved by a CRISPR system, including but not limited to gene editing, DNA cleavage, DNA nicking, DNA binding, regulation of gene expression, CRISPR activation (CRISPRa), CRISPR interference (CRISPRi), and any other function that can be achieved by linking a cas protein to another effector, thereby achieving the effector function on a target sequence recognized by the cas protein.
  • CRISPRa CRISPR activation
  • CRISPRi CRISPR interference
  • a nuclease-free cas protein can be fused to a transcription factor, a deaminase, a methylase, etc.
  • the resulting fusion protein in the presence of a guide RNA for the target, can be used to regulate the transcription of, deaminate, or methylate, the target.
  • portion refers to any portion of the sequence (e.g., a nucleotide subsequence or an amino acid subsequence) that is smaller than the complete sequence.
  • Portions of polynucleotides can be any length, for example, at least 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 150, 200, 300 or 500 or more nucleotides in length.
  • a portion of a guide sequence can be about 50%, 40%, 30%, 20%, 10% of the guide sequence, e.g., one-third of the guide sequence or shorter, e.g., 7, 6, 5, 4, 3, or 2 nucleotides in length.
  • a Cas9 single-mutant nickase and a Cas9 double-mutant null-nuclease are derived from a wild-type Cas9 protein.
  • substantially identical in the context of two or more polynucleotides (or two or more polypeptides) refers to sequences or subsequences that have at least about 60%, at least about 70%, at least about 80%, at least about 90%, about 90-95%, at least about 95%, at least about 98%, at least about 99% or more nucleotide (or amino acid) sequence identity, when compared and aligned for maximum correspondence using a sequence comparison algorithm or by visual inspection.
  • the “substantial identity” between polynucleotides exists over a region of the polynucleotide at least about 20 nucleotides in length, at least about 50 nucleotides in length, at least about 100 nucleotides in length, at least about 200 nucleotides in length, at least about 300 nucleotides in length, at least about 500 nucleotides in length, or over the entire length of the polynucleotide.
  • the “substantial identity” between polypeptides exists over a region of the polypeptide at least about 50 amino acid residues in length, at least about 100 amino acid residues in length, or over the entire length of the polypeptide.
  • FIG. 1 Shown in FIG. 1 is a diagram of CRISPR-Cas9-mediated sequence-specific cleavage of DNA.
  • the guide RNA is depicted as sgRNA with an exemplary 20-nucleotide (or 20-nt; nucleotide is often abbreviated as “nt”) guide sequence (other guide sequences may be, for example, from about 15 to about 30 nts in length) within the 5′ domain, an internally positioned base-paired stem, and a 3′ domain.
  • the guide sequence is complementary to an exemplary 20-nt target sequence in a DNA target.
  • the stem corresponds to a repeat sequence in crRNA and is complementary to a sequence in the tracrRNA.
  • the 3′ domain of the guide RNA corresponds to the 3′ domain of the tracrRNA that binds a Cas9 nuclease.
  • the Cas9:gRNA complex binds and cleaves a target DNA sequence or protospacer directly upstream of a PAM sequence recognized by Cas9.
  • a 3-nt PAM sequence is exemplified; however, other PAM sequences including 4-nt, 5-nt and even longer PAM sequences are known.
  • Guide RNAs for CRISPR-Cas genome editing, function in RNA-protein complexes where the RNA acts both as a scaffold for the protein and as sequence recognition for the duplex DNA target.
  • the complex recognizes genomic DNA through first scanning for the nucleotide PAM sequence. Once a PAM sequence is identified the RNA-protein complex attempts to form an RNA/DNA duplex between the genomic DNA target and the guide sequence of the guide RNA. This duplex is first initiated by a Cas protein-mediated base pairing of the “seed region” of the gRNA, in which the seed region is thought to be around ten nucleotides in length.
  • RNA/DNA duplex is formed by the hybridization of the remaining nucleotides on the 5′ end of the guide RNA; this typically results in a 20-nucleotide DNA/RNA duplex and proceeds to a double-stranded cleavage of the target DNA by the protein complex.
  • CRISPR-Cas RNA-protein complexes cleave genomic DNA as a first step in the process to inactivate or modify a gene through repair or recombination. In this process the cleavage of genomic DNA at unintentional “off-target” sites can have unwanted consequences, such as the creation of sequence mutations elsewhere in the genome. Currently, these off-target cleavage events are either being detected by screening techniques, removed by breeding techniques, or ignored.
  • CRISPR-Cas RNA-protein complexes evolved as an adaptive immune system in prokaryotes; an improvement in sequence specificity of these complexes would constitute a significant advance and innovation toward CRISPR-Cas RNA-protein complexes having wide utility as a tool in eukaryotic genomics.
  • Sequence specificity starts by a gRNA:Cas protein complex being able to scan, detect, and bind to a target site, or string of contiguous nucleotides within the entire genome of an organism.
  • the target sequence needs to be long enough that its string or sequence of contiguous nucleotides exists only once in the entire genome of the organism of interest and is located at the site of the desired genome editing.
  • the length of the string of contiguous nucleotides, or polynucleotide, necessary to impart uniqueness within a genome is determined by the “information content” of that particular polynucleotide.
  • the target polynucleotide needs to be in the range of 18 to 22 nucleotides in length to have enough information content to be unique (J. Mol. Biol. (1986), 188, 415-431) in the entire genome.
  • the longer the target polynucleotide the more information content and the more likely its sequence will exist only once in a genome a 19-nucleotide sequence has more information content than an 18-nucleotide sequence
  • a 20-nucleotide sequence has more information content than a 19-nucleotide sequence and so on.
  • a unique 20-nucleotide sequence could match the sequence of all but 1 nucleotide in a different 20-nt sequence elsewhere in the genome, and the sequence containing a single mismatch comprises an off-target site.
  • the relative binding of a guide sequence to the off-target sequence versus the 20-nucleotide target sequence is controlled by the respective binding energies and kinetic equilibria between the guide sequence and the on-target sequence as well as the off-target sequence.
  • the differential binding energies of oligonucleotides are controlled and can be maximized by the cooperative effect of DNA and RNA binding.
  • Cooperativity has been defined for DNA and RNA binding during hybridization in two ways. First, when an oligonucleotide begins to bind its individual nucleotide subunits, the binding of nucleotide subunits to complementary nucleobases has a positive effect on the subsequent binding of the next adjacent nucleotide in the oligonucleotide sequence. At the same time the unbinding of an individual nucleotide has a negative effect on the binding of the next adjacent nucleotide.
  • the unbinding of that mismatched pair has a negative effect on the binding of the adjacent nucleotide pair and likewise when a matched nucleotide pair binds successfully it has a positive effect on the binding of the adjacent nucleotide pair.
  • the intermediate states are statistically underrepresented relative to a system where the steps occur independently of each other. In other words, there are limited degrees of freedom and a limited number of kinetic states other than bound or unbound.
  • DNA oligonucleotides comprising 14 nucleotides in length were constructed from two oligonucleotides, each 7 nucleotides in length, which were covalently connected by 1,3-diaminopropane or 1,3-propanediol linkages by using chemical ligation.
  • the flexible oligonucleotide was bound to a complementary 14-nucleotide DNA oligonucleotide and the binding energy measured. Without the rigidity of the natural DNA backbone the flexible 14-nucleotide DNA single strand bound with a significantly lower binding energy, as if it were two independent 7-nucleotide DNA oligonucleotides lacking cooperativity.
  • cooperativity allows a higher degree of match verses mismatch distinction than would be seen in a non-cooperative system and is an important component for increasing the specificity of nucleotide sequences.
  • the Cas protein has been shown by crystal structure to pre-order the 10 nucleotides of the seed region into a single-stranded portion of an A-form helix.
  • the preordering of the RNA into an A-form helix limits the number of kinetic states that the guide sequence of the gRNA can adopt, thus increasing the cooperativity of the DNA/RNA hybridization in the seed region.
  • the overall binding energy of a string of nucleotides to a complementary string of nucleotides is typically defined by the melting temperature (Tm).
  • Tm melting temperature
  • the melting temperature is the temperature required to dissociate two bound nucleotide strings or strands (i.e., bound by base pairing or hybridization) to the point that they are 50% bound and 50% unbound.
  • the melting temperature is measured by a phenomenon known as hyperchromicity.
  • the UV absorption is increased when the two bound oligonucleotide strands are being separated by heat. Heat denaturation of oligonucleotides causes the double helix structure to unwind to form single-stranded oligonucleotides.
  • the duplex When two bound oligonucleotides in solution are heated above their melting temperature, the duplex unwinds to form two single strands that absorb more light than the duplex.
  • the UV absorbance is graphed as a function of the temperature, a sigmoidal curve is obtained at the point where the duplex begins to dissociate and the center of the sigmoidal curve is defined as the Tm.
  • FIG. 4 is a graph demonstrating how UV absorbance increases as an oligonucleotide duplex separates into separate strands by heating.
  • a sigmoidal curve reflects the dissociate of the duplex into separate strands, and the center of the sigmoidal curve is the T m of the duplex. This curve indicates that the melting temperature of a 20-nucleotide DNA/RNA duplex at physiological salt concentrations is around 50° C.
  • An oligonucleotide duplex has the greatest match verses mismatch specificity at the melting temperature where only 50% of the duplex is formed. At this temperature a single mismatch at an off-target polynucleotide will block hybridization of the oligonucleotide if the binding and unbinding of the duplex has a high degree of cooperativity or shows a steep sigmoidal curve. If a gene editing experiment is performed at 37° C., then the best discrimination of match verses mismatch would be obtained using a guide RNA whose binding to its target would have a Tm of 37° C.
  • truncation of an RNA in a RNA/DNA duplex decreases the Tm of that duplex by about 2° C. per base pair.
  • Truncation of the guide sequence in a gRNA to 17 nucleotides would decrease the binding energy by around 6° C. resulting in a duplex Tm of around 42° C.
  • significant information content is lost such that it can more readily find an increased number of off-target sites identical or similar to the 17-nt guide sequence across the entire genome.
  • a more useful approach would be to decrease the binding energy of the 20-nucleotide RNA/DNA duplex through chemical modification while retaining cooperativity of binding and unbinding.
  • FIG. 5A also illustrates a single-guide RNA or two-piece dual-guide RNA 501 (wherein a crRNA segment and a tracrRNA segment form a hybridized duplex). Moving from left to right (i.e., from the 5′ end to the 3′ end of the guide RNA), FIG.
  • 5A generally shows an extension 503 (sometimes referred to as an overhang) on the guide RNA, a sampling and locking region 505 , a Cas protein-binding seed region 507 (typically a 10-nt portion), a dual-guide stem 509 or a single-guide stem-loop 511 , and a tracrRNA region 513 .
  • Cas9 protein can bind any or all of these gRNA regions except perhaps 503 .
  • the guide sequence comprises the locking, sampling, and seed regions.
  • the guide sequence of about 20 nucleotides on the 5′ end of the guide RNA is what binds and forms a stable duplex with the target DNA. This hybridization binding relative to competing hybridization with other sites of similar sequence determines the overall specificity of the gRNA for the target and thus the specificity of genome editing or gene inactivation by a gRNA:Cas protein complex.
  • the binding of guide RNAs to their target polynucleotides occurs via Cas9-mediated formation of a seed RNA/DNA duplex initiated by the 3′ end of the guide sequence. Once the initial seed duplex is formed, the gRNA should continue to hybridize toward its 5′ end like a zipper.
  • FIG. 5B illustrates how, after the initial formation of a seed duplex 517 from gRNA 501 and genomic DNA 515 , the binding of the nucleotides proceeds sequentially through the sampling region.
  • the sampling region is the region adjacent to the seed region, and the hybridization binding of these nucleotides proceeds until the point where the binding energy of the duplex is approximately equivalent to the temperature at which the binding is occurring.
  • the rate of binding and unbinding in this region is controlled by the larger equilibrium of the bound versus unbound RNA (e.g., 501 versus 519 or 501 versus 521 ) which is also controlled by interaction of the Cas9 protein.
  • the typical sampling region spans the 5 to 6 nucleotides just 5′ of the 10-nucleotide seed region, based on the fact that a 15- to 16-nucleotide RNA/DNA duplex has a Tm of approximately 37° C.
  • the equilibrium between bound RNA versus unbound RNA changes such that its unbinding or release of a target polynucleotide is negatively impacted by the overall equilibrium of the bound versus unbound RNA which now lies significantly in the direction of the bound or hybridized state.
  • the effect that the larger equilibrium of the bound versus unbound RNA has on specificity was shown by Slaymaker et al. (2016), “Rationally engineered Cas9 nucleases with improved specificity”, Science 351, 84-8. Slaymaker et al.
  • a Cas9 protein was engineered that decreased off-target indel formation by converting positively charged amino acids in the nucleic acid-binding groove (or nt groove) of the protein to neutrally charged alanine residues. These changes decrease the overall Cas9-mediated affinity of the guide RNA for the double-stranded target DNA and force the affinity to depend more on the RNA/DNA hybridization including base-pair recognition.
  • FIG. 5C illustrates how chemical modifications of nucleotides that decrease the binding energy of individual base pairs yet retain a high level of cooperativity can be utilized in the seed and sampling regions of a gRNA 525 to extend the sampling region beyond 5 or 6 nucleotides.
  • This effect can significantly increase the specificity of binding of a guide RNA 525 to genomic DNA 515 , as the chemical modifications increase the number of nucleotides required to achieve a hybridization binding energy equivalent to the temperature at which the binding is occurring.
  • the overall number of nucleotides required to shift the larger equilibrium of the bound versus unbound RNA is increased, and this is calculated to result in an overall increase in sequence specificity.
  • nucleotide modifications can be used to decrease the activity of gRNA:Cas protein complexes toward partially complementary off-target polynucleotides through any of three different motifs in the guide sequence portion: the heterocyclic nucleobase, the sugar, and the internucleotide phosphate linkage.
  • the modification does not significantly increase non-specific binding or significantly decrease the cooperativity of hybridization, as either or both can promote off-target binding, nicking or cleaving by the Cas protein, which is undesirable.
  • nucleobases in a guide sequence it is possible to decrease the number of atoms accessible for base pairing and thus decrease the hydrogen-bonding potential that drives hybridization.
  • decreasing the hydrogen bonding potential can also increase recognition of off-target polynucleotide sequences through alternative base pairing.
  • An example of modifying the nucleobase to promote high specificity is to install a 2-thiouridine in place of a uridine in a guide sequence.
  • Uridine nucleotides which normally bind to adenosine nucleotides by two hydrogen bonds, can alternatively bind to guanosine nucleotides to yield a rather weak base pair often referred to as a G-U wobble pair. If 2-thiouridine is used instead of uridine, the 2-thiouridine can form only a less stable G-U wobble pair if any, because the sulfur substituent on the C2 position of the uracil base cannot serve as a hydrogen bond acceptor.
  • This same strategy can be used to decrease the hydrogen bonding potential of a typical Guanosine/Cytidine base pair by reducing the number of potential hydrogen bonds from three to two, for example by using either 2-thiocytidine or 4-thioguanosine. Their diminished potential for forming hydrogen bonds in modified G-C base pairs is illustrated here.
  • Modifications of the sugar moiety of a ribonucleotide can also be used to decrease the affinity of a guide sequence to a complementary DNA strand. This can be done in two ways, the first is to alter the sugar pucker, and the second is to deform the RNA/DNA duplex by steric crowding. Generally, the sugar pucker is described to be in either one of two states: the 2′-endo (south, DNA-like) sugar pucker, or the 3′-endo (north, RNA-like) sugar pucker.
  • the 2′-endo pucker is thought to have a destabilizing effect on base pairing, and this is thought result from changes in the torsional angle of the glycosidic bond, thus preventing formation of the highest-affinity base-stacking geometry.
  • modifications to RNA that can result in a 2′-endo conformation are deoxyribose, 2′-deoxy-2′-fluoroarabinofuranosyl, 2′-deoxy-2′-fluororibofuranosyl, 2′-O-phenyl, 2′-thiophenyl, 2′-S-thiophenyl, 2′-methyl, 2′-ethyl, 2′-propyl, 2′-allyl, 2′-allylphenyl, 2′-methylhydroxy, 2′-methyloxymethyl, 2′-O-carbamate, 2′-O-ethylamino, 2′-O-allylamino, 2′-O-propylamino and 2′-O-substituted
  • Modification of an internucleotide bond can also decrease the binding affinity of nucleotides while maintaining the cooperativity of base pair hybridization.
  • examples of these are phosphonoacetates, thiophosphonoacetates, phosphonocarboxylates, thiophosphonocarboxylates, phosphonopropionates, phosphonothiopropionates, methylphosphonates, methylphosphonothioates, and boranophosphonates.
  • a sugar modification or nucleobase modification that increases or decreases the binding energy of the overall guide RNA can be added to modulate or further tune the binding energy from incorporation of other modifications to the guide RNA.
  • incorporation of a 2′-O-methyl-thiophosphonoacetate (MSP) will decrease the binding energy of the guide RNA by ⁇ 1.5 degrees as compared to the unmodified guide RNA. If a 2′-O-methyl nucleotide is incorporated elsewhere in the guide sequence it will increase the overall binding energy by ⁇ 0.2 degrees and the resulting guide RNA will have a decreased overall binding energy of ⁇ 1.3 degrees as compared to the unmodified guide RNA.
  • MSP 2′-O-methyl-thiophosphonoacetate
  • the sugar modification comprises 2′-O—C 1-3 alkyl-O—C 1-3 alkyl, such as 2′-methoxyethoxy (2′-O—CH 2 CH 2 OCH 3 ) also known as 2′-O-(2-methoxyethyl) or 2′-MOE.
  • the sugar modification comprises 2′-halo, such as 2′-F, 2′-Br, 2′-Cl, or 2′-I.
  • the sugar modification comprises 2′-NH 2 .
  • the sugar modification comprises 2′-H (e.g., a 2′-deoxynucleotide).
  • the sugar modification comprises 2′-arabino or 2′-F-arabino. In certain embodiments, the sugar modification comprises 2′-LNA or 2′-ULNA. In certain embodiments, the sugar comprises a 4′-thioribosyl.
  • a nucleotide sugar modification or nucleobase modification that increases or decreases the binding energy of the overall guide RNA can be incorporated on the same nucleotide where a phosphodiester linkage is modified to modulate the binding energy of the modified nucleotide.
  • a phosphodiester linkage is modified to modulate the binding energy of the modified nucleotide.
  • 3′-phosphonocarboxylate linkages can be used with sugar modifications such as 2′-O-methyl, 2′-F, or 2′-O-(2-methoxyethyl).
  • the sugar comprises 2′-O—C 1-3 alkyl-O—C 1-3 alkyl, such as 2′-methoxyethoxy (2′-O—CH 2 CH 2 OCH 3 ) also known as 2′-O-(2-methoxyethyl) or 2′-MOE.
  • the sugar comprises 2′-halo, such as 2′-F, 2′-Br, 2′-Cl, or 2′-I.
  • the sugar comprises 2′-NH 2 .
  • the sugar comprises 2′-H (e.g., a 2′-deoxynucleotide).
  • the sugar comprises 2′-arabino or 2′-F-arabino.
  • the sugar comprises 2′-LNA or 2′-ULNA.
  • the sugar comprises a 4′-thioribosyl.
  • the present invention comprises a chemically modified guide RNA that has guide RNA functionality.
  • the chemically modified guide RNA comprises at least one specificity-enhancing modification and may comprise other chemical modifications having more functions or different functions than specificity enhancement.
  • a guide RNA that comprises any backbone or internucleotide linkage other than a natural phosphodiester internucleotide linkage possesses a chemical modification and therefore is a chemically modified guide RNA.
  • the retained functionality includes binding a Cas protein.
  • the retained functionality includes binding a target polynucleotide.
  • the retained functionality includes targeting a Cas protein or a gRNA:Cas protein complex to a target polynucleotide. In certain embodiments, the retained functionality includes nicking a target polynucleotide by a gRNA:Cas protein complex. In certain embodiments, the retained functionality includes cleaving a target polynucleotide by a gRNA:Cas protein complex. In certain embodiments, the retained functionality is any other known function of a guide RNA in a CRISPR-Cas system with a Cas protein, including an artificial CRISPR-Cas system with an engineered Cas protein. In certain embodiments, the retained functionality is any other function of a natural guide RNA.
  • the specificity-enhancing modification is a deoxyribose nucleotide, a 2′-deoxy-2′-fluoroarabinofuranosyl nucleotide, a 2′-deoxy-2′-fluororibofuranosyl nucleotide, a sugar having a 2′-O-phenyl, 2′-S-thiophenyl, 2′-methyl, 2′-ethyl, 2′-propyl, 2′-allyl, 2′-allylphenyl, 2′-methylhydroxy, 2′-methyloxymethyl, 2′-O-carbamate, 2′-O-ethylamino, 2′-O-allylamino, 2′-O-propylamino, or 2′-O-substituted phenyl, or combinations thereof.
  • the specificity-enhancing modification is a phosphonoacetate, thiophosphonoacetate, phosphonopropionate, phosphonothiopropionate, methylphosphonate, methylphosphonothioate, or boranophosphonate; or combinations of any of the foregoing.
  • a nucleotide sugar modification incorporated into the guide RNA is selected from the group consisting of deoxyribose, 2′-deoxy-2′-fluoroarabinofuranosyl, 2′-deoxy-2′-fluororibofuranosyl, and sugars having 2′-O-phenyl, 2′-S-thiophenyl, 2′-methyl, 2′-ethyl, 2′-propyl, 2′-allyl, 2′-allylphenyl, 2′-methylhydroxy, 2′-methyloxymethyl, 2′-O-carbamate, 2′-O-ethylamino, 2′-O-allylamino, 2′-O-propylamino, and 2′-O-substituted phenyl.
  • an internucleotide linkage modification incorporated into the guide RNA is selected from the group consisting of: phosphorothioate “P(S)” (P(S)), phosphonocarboxylate (P(CH 2 ) n COOR) such as phosphonoacetate “PACE” (P(CH 2 COO ⁇ )), thiophosphonocarboxylate ((S)P(CH 2 ) n COOR) such as thiophosphonoacetate “thioPACE” ((S)P(CH 2 COO ⁇ )), alkylphosphonate (P(C 1-3 alkyl) such as methylphosphonate —P(CH 3 ), boranophosphonate (P(BH 3 )), and phosphorodithioate (P(S) 2 ).
  • P(S) phosphorothioate
  • P(CH 2 ) n COOR such as phosphonoacetate “PACE” (P(CH 2 COO ⁇ )
  • an internucleotide linkage modification incorporated into the guide RNA is selected from the group consisting of phosphonoacetates, thiophosphonoacetates, phosphonopropionates, phosphonothiopropionates, methylphosphonates, methylphosphonothioates, and boranophosphonates.
  • a nucleobase (“base”) modification incorporated into the guide RNA is selected from the group consisting of: 2-thiouracil (“2-thioU”), 2-thiocytosine (“2-thioC”), 4-thiouracil (“4-thioU”), 6-thioguanine (“6-thioG”), 2-aminopurine, pseudouracil, hypoxanthine, 7-deazaguanine, 7-deaza-8-azaguanine, 7-deazaadenine, 7-deaza-8-azaadenine, 5-methylcytosine (“5-methylC”), 5-methyluracil (“5-methylU”), 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5,6-dehydrouracil, 5-ethynylcytosine, 5-aminoallyluracil (“5-aminoallylU”), 5-aminoallyl-cytosine (“5-aminoallylC”), an abasic nucleotide, Unstructured Nucleic
  • one or more isotopic modifications are introduced on the nucleotide sugar, the nucleobase, the phosphodiester linkage and/or the nucleotide phosphates.
  • modifications include nucleotides comprising one or more 15 N, 13 C, 14 C, Deuterium, 3 H, 32 P, 131 I atoms or other atoms or elements used as tracers.
  • an “end” modification incorporated into the guide RNA is selected from the group consisting of: PEG (polyethyleneglycol), hydrocarbon linkers (including: heteroatom (O,S,N)-substituted hydrocarbon spacers; halo-substituted hydrocarbon spacers; keto-, carboxyl-, amido-, thionyl-, carbamoyl-, thionocarbamaoyl-containing hydrocarbon spacers), spermine linkers, dyes including fluorescent dyes (for example fluoresceins, rhodamines, cyanines) attached to linkers such as for example 6-fluorescein-hexyl, quenchers (for example dabcyl, BHQ) and other labels (for example biotin, digoxigenin, acridine, streptavidin, avidin, peptides and/or proteins).
  • PEG polyethyleneglycol
  • hydrocarbon linkers including: heteroatom (O,S,N)-substitute
  • an “end” modification comprises a conjugation (or ligation) of the guide RNA to another molecule comprising an oligonucleotide (comprising deoxynucleotides and/or ribonucleotides), a peptide, a protein, a sugar, an oligosaccharide, a steroid, a lipid, a folic acid, a vitamin and/or other molecule.
  • an oligonucleotide comprising deoxynucleotides and/or ribonucleotides
  • an “end” modification incorporated into the guide RNA is located internally in the guide RNA sequence via a linker such as for example 2-(4-butylamidofluorescein)propane-1,3-diol bis(phosphodiester) linker (depicted below), which is incorporated as a phosphodiester linkage and can be incorporated anywhere between two nucleotides in the guide RNA.
  • a linker such as for example 2-(4-butylamidofluorescein)propane-1,3-diol bis(phosphodiester) linker (depicted below), which is incorporated as a phosphodiester linkage and can be incorporated anywhere between two nucleotides in the guide RNA.
  • linkers include for example by way of illustration, but are not limited to:
  • the end modification comprises a terminal functional group such as an amine, a thiol (or sulfhydryl), a hydroxyl, a carboxyl, carbonyl, thionyl, thiocarbonyl, a carbamoyl, a thiocarbamoyl, a phosphoryl, an alkene, an alkyne, an halogen or a functional group-terminated linker, either of which can be subsequently conjugated to a desired moiety, for example a fluorescent dye or a non-fluorescent label or tag or any other molecule such as for example an oligonucleotide (comprising deoxynucleotides and/or ribonucleotides, including an aptamer), an amino acid, a peptide, a protein, a sugar, an oligosaccharide, a steroid, a lipid, a folic acid, a vitamin.
  • the conjugation employs standard
  • the label or dye is attached or conjugated to a modified nucleotide in the gRNA.
  • a fluorescent dye or other moiety such as a non-fluorescent label or tag (for example biotin, avidin, streptavidin, or moiety containing an isotopic label such as 15 N, 13 C, 14 C, Deuterium, 3 H, 32 P, 125 I and the like) or any other molecule such as for example an oligonucleotide (comprising deoxynucleotides and/or ribonucleotides including an aptamer), an amino acid, a peptide, a protein, a sugar, an oligosaccharide, a steroid, a lipid, a folic acid, a vitamin or other molecule can be effectuated using the so-called “click” chemistry or the so-called “squarate” conjugation chemistry.
  • the “click” chemistry refers to the [3+2] cyclo
  • the conjugation can be effectuated by alternative cycloaddition such as Diels-Alder [4+2] cycloaddition of a n-conjugated diene moiety with an alkene moiety.
  • alternative cycloaddition such as Diels-Alder [4+2] cycloaddition of a n-conjugated diene moiety with an alkene moiety.
  • the “squarate” conjugation chemistry links two moieties each having an amine via a squarate derivative to result in a squarate conjugate that contains a squarate moiety (see e.g., Tietze et al. (1991) Chem. Ber., 124, 1215-21, the contents of which are hereby incorporated by reference in their entirety).
  • a fluorescein containing a linker amine is conjugated to an oligoribonucleotide containing an amine through a squarate linker as described in the scheme below.
  • An example of the squarate linker is depicted in the following scheme:
  • the present technology provides a guide RNA having at least one specificity-enhancing modification, constituting a modified gRNA and optionally a stability-enhancing modification.
  • At least one specificity-enhancing modification is within the guide sequence or crRNA segment of the guide RNA.
  • the modification is within the guide sequence of the crRNA.
  • the modification is within the first five (5) nucleotides of the 5′ end of the guide sequence or crRNA segment.
  • the modification is within the first four (4) nucleotides of the guide sequence or crRNA segment.
  • the modification is within the first three (3) nucleotides of the guide sequence or crRNA segment.
  • the modification is also within a 5′-overhang on the crRNA segment.
  • At least one specificity-enhancing modification is within nucleotides 4-N to 20-N, alternatively within nucleotides 5-N to 20-N, alternatively within nucleotides 10-N to 20-N, alternatively within nucleotides 13-N to 20-N, alternatively within nucleotides 13-N to 14-N or 16-N to 19-N, alternatively within nucleotides 13-N to 14-N or 16-N to 18-N.
  • the modification is at nucleotides 4-N, 5-N, 7-N, 9-N, 10-N, 11-N, or any combination thereof.
  • a modified gRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 specificity-enhancing modified nucleotides in the guide sequence portion of the gRNA and up to 100 additional modified nucleotides in the other portions or segments of the gRNA.
  • the modified gRNA comprises a 5′ extension or overhang on the guide sequence portion, and the extension is 1 to 20 nucleotides in length comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 specificity-enhancing modified nucleotides in addition to the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 specificity-enhancing modified nucleotides in the guide sequence portion of the gRNA and optionally comprising up to 100 additional modified nucleotides in the other portions of the gRNA.
  • all nucleotides in a gRNA are modified. In certain embodiments, all the modifications are the same.
  • the modified gRNA comprises a combination of differently modified nucleotides. In certain embodiments, the modified gRNA comprises two or more modified nucleotides. In certain embodiments, the modified gRNA comprises three or more modified nucleotides. In certain embodiments, the modified nucleotides are arranged contiguously. In certain embodiments, the modified gRNA comprises at least one contiguous stretch of modified nucleotides.
  • a chemical modification is within the 5′ portion of the guide RNA.
  • a chemical modification within a 5′ portion refers to a modification within the 5′ portion of the crRNA segment of the guide RNA, and not to a modification within a 5′ portion of a tracrRNA segment.
  • a guide RNA is a single guide RNA, it has one 5′ portion, located in the crRNA segment.
  • the modification is within the first five (5) nucleotides of the 5′ portion of the guide RNA.
  • the modification is within the first three (3) nucleotides of the 5′ portion of the guide RNA.
  • a chemical modification is incorporated in the 5′ portion or the 3′ portion of the guide RNA, particularly within the first 5 or 10 nucleotides of the 5′ portion or within the last 5 or 10 nucleotides of the 3′ portion to, for example, protect the RNA from degradation by nucleases or for other purposes.
  • the modification is in both the 5′ portion and the 3′ portion of the guide RNA, particularly within the first 5 or 10 nucleotides of the 5′ portion and within the last 5 or 10 nucleotides of the 3′ portion to, for example, protect the RNA from degradation by nucleases or for other purposes.
  • a guide RNA comprises 40 or fewer, alternatively 20 or fewer, alternatively 15 or fewer, alternatively 10 or fewer, alternatively 5 or fewer, alternatively 3 or fewer deoxyribonucleotide residues in the 5′ or 3′ portion of the guide RNA.
  • each RNA molecule may comprise modification(s) at the 5′-end, 3′-end, or both.
  • consecutive nucleotides at the end (5′, 3′, or both) are modified, such as 2, 3, 4, 5 or more consecutive nucleotides.
  • the guide sequence consists of 20-N nucleotides, where N is an integer between ⁇ 10 and 10 (optionally between ⁇ 10 and 6). N can be selected from ⁇ 10, ⁇ 9, ⁇ 8, ⁇ 7, ⁇ 6, ⁇ 5, ⁇ 4, ⁇ 3, ⁇ 2, ⁇ 1, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.
  • the guide sequence comprises at least one specificity-enhancing modification at nucleotide position (starting from the 5′-end of the guide sequence) 4-N, 5-N, 7-N, 9-N or 11-N, or a combination thereof. A few examples are described below.
  • the guide sequence consists of nucleotides 1 through 20, counted from the 5′ end of the guide sequence, and comprises a chemical modification at at least one nucleotide selected from positions 4, 5, 7, 9, 10, and 11.
  • the chemical modification is at nucleotide 11 of the guide sequence.
  • the chemical modification is at nucleotide 5 of the guide sequence.
  • the chemical modification is at nucleotide 7 of the guide sequence.
  • the chemical modification is at nucleotide 10 of the guide sequence.
  • the chemical modification is at nucleotide 9 of the guide sequence.
  • the chemical modification is at nucleotide 4 of the guide sequence.
  • the guide RNA comprises at least one end modification.
  • the guide RNA comprises a 5′ extension or overhang upstream of the guide sequence.
  • the guide sequence consists of nucleotides 1 through 19, counted from the 5′ end of the guide sequence, and comprises a chemical modification at at least one nucleotide selected from positions 3, 4, 6, 8, 9, and 10.
  • the chemical modification is at nucleotide 4 of the guide sequence.
  • the chemical modification is at nucleotide 6 of the guide sequence.
  • the chemical modification is at nucleotide 8 of the guide sequence.
  • the chemical modification is at nucleotide 9 of the guide sequence.
  • the chemical modification is at nucleotide 10 of the guide sequence.
  • the chemical modification is at nucleotide 3 of the guide sequence.
  • the guide RNA comprises at least one end modification.
  • the guide RNA comprises a 5′ extension or overhang upstream of the guide sequence.
  • the guide sequence consists of nucleotides 1 through 17, counted from the 5′ end of the guide sequence, and comprises a chemical modification of at least one nucleotide selected from positions 1, 2, 4, 6, 7, and 8.
  • the chemical modification is at nucleotide 2 of the guide sequence.
  • the chemical modification is at nucleotide 4 of the guide sequence.
  • the chemical modification is at nucleotide 6 of the guide sequence.
  • the chemical modification is at nucleotide 7 of the guide sequence.
  • the chemical modification is at nucleotide 8 of the guide sequence.
  • the chemical modification is at nucleotide 1 of the guide sequence.
  • the guide RNA comprises at least one end modification.
  • the guide RNA comprises a 5′ extension or overhang upstream of the guide sequence.
  • the guide sequence consists of nucleotides 1 through 16, counted from the 5′ end of the guide sequence, and comprises a chemical modification of at least one nucleotide selected from positions 1, 3, 5, 6, and 7.
  • the chemical modification is at nucleotide 1 of the guide sequence.
  • the chemical modification is at nucleotide 3 of the guide sequence.
  • the chemical modification is at nucleotide 5 of the guide sequence.
  • the chemical modification is at nucleotide 6 of the guide sequence.
  • the chemical modification is at nucleotide 7 of the guide sequence.
  • the guide RNA comprises at least one end modification.
  • the guide RNA comprises a 5′ extension or overhang upstream of the guide sequence.
  • the guide sequence consists of nucleotides 1 through 20, counted from the 5′ end of the guide sequence, and comprises at least two chemical modifications at nucleotides selected from positions 5, 6, 7, 8, 9, 10, 16, and 17.
  • the chemical modification is at nucleotide 6 and nucleotide 10 of the guide sequence.
  • the chemical modification is at nucleotide 5 and nucleotide 17 of the guide sequence.
  • the chemical modification is at nucleotide 6 and nucleotide 7 of the guide sequence.
  • the chemical modification is at nucleotide 10 and nucleotide 17 of the guide sequence.
  • the chemical modification is at nucleotide 5 and nucleotide 16 of the guide sequence. In certain embodiments, the chemical modification is at nucleotide 10 and nucleotide 16 of the guide sequence. In certain embodiments, the chemical modification is at nucleotide 5 and nucleotide 9 of the guide sequence. In certain embodiments, the chemical modification is at nucleotide 9 and nucleotide 16 of the guide sequence. In certain embodiments, the chemical modification is at nucleotide 8 and nucleotide 17 of the guide sequence. In certain embodiments, the guide RNA comprises at least one end modification. In certain embodiments, the guide RNA comprises a 5′ extension or overhang upstream of the guide sequence.
  • the guide sequence consists of nucleotides 1 through 19, counted from the 5′ end of the guide sequence, and comprises at least two chemical modifications at nucleotides selected from positions 4, 5, 6, 7, 8, 9, 15, and 16.
  • the chemical modification is at nucleotide 5 and nucleotide 9 of the guide sequence.
  • the chemical modification is at nucleotide 4 and nucleotide 16 of the guide sequence.
  • the chemical modification is at nucleotide 5 and nucleotide 6 of the guide sequence.
  • the chemical modification is at nucleotide 9 and nucleotide 16 of the guide sequence.
  • the chemical modification is at nucleotide 4 and nucleotide 15 of the guide sequence. In certain embodiments, the chemical modification is at nucleotide 9 and nucleotide 15 of the guide sequence. In certain embodiments, the chemical modification is at nucleotide 4 and nucleotide 8 of the guide sequence. In certain embodiments, the chemical modification is at nucleotide 8 and nucleotide 15 of the guide sequence. In certain embodiments, the chemical modification is at nucleotide 7 and nucleotide 16 of the guide sequence.
  • the chemical modification is at nucleotide 3 and nucleotide 14 of the guide sequence. In certain embodiments, the chemical modification is at nucleotide 8 and nucleotide 14 of the guide sequence. In certain embodiments, the chemical modification is at nucleotide 3 and nucleotide 7 of the guide sequence. In certain embodiments, the chemical modification is at nucleotide 7 and nucleotide 14 of the guide sequence. In certain embodiments, the chemical modification is at nucleotide 6 and nucleotide 15 of the guide sequence.
  • the chemical modification is at nucleotide 2 and nucleotide 13 of the guide sequence. In certain embodiments, the chemical modification is at nucleotide 7 and nucleotide 13 of the guide sequence. In certain embodiments, the chemical modification is at nucleotide 2 and nucleotide 6 of the guide sequence. In certain embodiments, the chemical modification is at nucleotide 6 and nucleotide 13 of the guide sequence. In certain embodiments, the chemical modification is at nucleotide 5 and nucleotide 14 of the guide sequence.
  • the guide sequence consists of nucleotides 1 through 16, counted from the 5′ end of the guide sequence, and comprises at least two chemical modifications at nucleotides selected from positions 1, 2, 3, 4, 5, 6, 12, and 13.
  • the chemical modification is at nucleotide 2 and nucleotide 6 of the guide sequence.
  • the chemical modification is at nucleotide 1 and nucleotide 13 of the guide sequence.
  • the chemical modification is at nucleotide 2 and nucleotide 3 of the guide sequence.
  • the chemical modification is at nucleotide 6 and nucleotide 13 of the guide sequence.
  • the guide sequence consists of nucleotides 1 through 15, counted from the 5′ end of the guide sequence, and comprises at least two chemical modifications at nucleotides selected from positions 1, 2, 3, 4, 5, 11, and 12.
  • the chemical modification is at nucleotide 1 and nucleotide 5 of the guide sequence.
  • the chemical modification is at nucleotide 1 and nucleotide 2 of the guide sequence.
  • the chemical modification is at nucleotide 5 and nucleotide 12 of the guide sequence.
  • the chemical modification is at nucleotide 5 and nucleotide 11 of the guide sequence.
  • the chemical modification is at nucleotide 4 and nucleotide 11 of the guide sequence.
  • the chemical modification is at nucleotide 3 and nucleotide 12 of the guide sequence.
  • a chemical modification comprises an end modification, such as a 5′ end modification or a 3′ end modification.
  • end modifications include, but are not limited to phosphorylation (as natural phosphate or polyphosphate or as modified phosphonate groups such as for example, alkylphosphonate, phosphonocarboxylate, phosphonoacetate, boranophosphonate, phosphorothioate, phosphorodithioate and the like), biotinylation, conjugating or conjugated molecules, linkers, dyes, labels, tags, functional groups (such as for example but not limited to 5′-amino, 5′-thio, 5′-amido, 5′carboxy and the like), inverted linkages, or hydrocarbon moieties which may comprise ether, polyethylene glycol (PEG), ester, hydroxyl, aryl, halo, phosphodiester, bicyclic, heterocyclic or other organic functional group.
  • the end modification comprises dimethoxytrityl
  • modified bases include, but are not limited to, synthetic and natural bases such as 2-thioU, 2-thioC, 4-thioU, 6-thioG, 2-aminoA, 2-aminopurine, pseudouracil, hypoxanthine, 7-deazaguanine, 7-deaza-8-azaguanine, 7-deazaadenine, 7-deaza-8-azaadenine, 5-methylC, 5-methylU, 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5,6-dehydrouracil, 5-propynylcytosine, 5-propynyluracil, 5-ethynylcytosine, 5-ethynyluracil, 5-allylU, 5-allylC, 5-aminoallyl-uracil, and 5-aminoallyl-cytosine.
  • synthetic and natural bases such as 2-thioU, 2-thioC, 4-thioU, 6-thioG, 2-aminoA, 2-aminopurine, pseudouracil, hypoxanthin
  • the modification comprises an abasic nucleotide.
  • the modification comprises a nonstandard purine or pyrimidine structure, such as Z or P, isoC or isoG, UNA, 5-methylpyrymidine, x(A,G,C,T,U) or y(A,G,C,T,U).
  • the modified gRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 modified bases.
  • the modified gRNA comprises at least 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130 or 140 modified bases. In certain embodiments, all bases in a gRNA are modified.
  • the modified gRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 modified sugars.
  • the modified gRNA comprises at least 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130 or 140 modified sugars. In certain embodiments, all sugars in a gRNA are modified.
  • the modification comprises a modified backbone (i.e., an internucleotide linkage other than a natural phosphodiester).
  • modified internucleotide linkages include, but are not limited to, a phosphorothioate internucleotide linkage, a chiral phosphorothioate internucleotide linkage, a phosphorodithioate internucleotide linkage, a boranophosphonate internucleotide linkage, a C 1-4 alkyl phosphonate internucleotide linkage such as a methylphosphonate internucleotide linkage, a boranophosphonate internucleotide linkage, a phosphonocarboxylate internucleotide linkage such as a phosphonoacetate internucleotide linkage, a phosphonocarboxylate ester internucleotide linkage such as a phosphonoacetate ester internucleotide
  • the modified gRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 modified internucleotide linkages.
  • the modified gRNA comprises at least 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130 or 140 modified internucleotide linkages. In certain embodiments, all internucleotide linkages in a gRNA are modified.
  • the modification is or comprises a 2′-O—C 1-4 alkyl, 2′-H, 2′-O—C 1-3 alkyl-O—C 1-3 alkyl, 2′-F, 2′-NH 2 , 2′-arabino, 2′-F-arabino, 2′-LNA, 2′-ULNA, 4′-thioribosyl, 2-thioU, 2-thioC, 4-thioU, 6-thioG, 2-aminoA, 2-aminopurine, pseudouracil, hypoxanthine, 7-deazaguanine, 7-deaza-8-azaguanine, 7-deazaadenine, 7-deaza-8-azaadenine, 5-MeC, 5-MeU, 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5,6-dehydrouracil, 5-propynylcytosine, 5-propynyluracil, 5-ethynylcytosine, 5-ethynyluracil, 5-
  • the modified nucleotide comprises a 2′-O-methyl-3′-phosphonoacetate. In certain embodiments, the modified nucleotide comprises a 2′-O-methyl-3′-phosphorothioate. In certain embodiments, the modified nucleotide comprises a 2′-O-methyl-3′-thiophosphonoacetate. In certain embodiments, the modified nucleotide comprises a 2′-O-methyl-3′-phosphonocarboxylate. In certain embodiments, the modified nucleotide comprises a 2′-deoxy-3′-phosphonoacetate. In certain embodiments, the modified nucleotide comprises a 2′-deoxy-3′-phosphorothioate.
  • the modified nucleotide comprises a 2′-deoxy-3′-thiophosphonoacetate. In certain embodiments, the modified nucleotide comprises a 2′-deoxy-3′-phosphonocarboxylate. In certain embodiments, the modified nucleotide comprises a 2′-halo-3′-phosphorothioate. In certain embodiments, the modified nucleotide comprises a 2′-halo-3′-phosphonoacetate. In certain embodiments, the modified nucleotide comprises a 2′-halo-3′-thiophosphonoacetate. In certain embodiments, the modified nucleotide comprises a 2′-halo-3′-phosphonocarboxylate.
  • the modified nucleotide comprises a 2′-fluoro-3′-phosphorothioate. In certain embodiments, the modified nucleotide comprises a 2′-fluoro-3′-phosphonoacetate. In certain embodiments, the modified nucleotide comprises a 2′-fluoro-3′-thiophosphonoacetate. In certain embodiments, the modified nucleotide comprises a 2′-fluoro-3′-phosphonocarboxylate. In certain embodiments, the modified nucleotide comprises a Z base. In certain embodiments, the modified nucleotide comprises a P base.
  • the guide RNA comprises an oligonucleotide represented by Formula (I): W—Y or Y—W (I)
  • W comprises a 2′-O—C 1-4 alkyl, 2′-H, 2′-O—C 1-3 alkyl-O—C 1-3 alkyl, 2′-F, 2′-NH 2 , 2′-arabino, 2′-F-arabino, 2′-LNA, 2′-ULNA, 4′-thioribosyl, 2-thioU, 2-thioC, 4-thioU, 6-thioG, 2-aminoA, 2-aminopurine, pseudouracil, hypoxanthine, 7-deazaguanine, 7-deaza-8-azaguanine, 7-deazaadenine, 7-deaza-8-azaadenine, 5-MeC, 5-MeU, 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5,6-dehydrouracil, 5-propynylcytosine, 5-propynyluracil, 5-ethynylcytosine, 5-ethynyluracil,
  • W comprises at least one contiguous stretch of modified nucleotides. In certain embodiments, W comprises a contiguous stretch of at least three (3) modified nucleotides. In certain embodiments, W comprises a contiguous stretch of at least four (4) modified nucleotides. In certain embodiments, W comprises a contiguous stretch of at least five (5) modified nucleotides.
  • the modification is a stability-altering modification.
  • Stability refers to the ability of the gRNA to resist degradation by enzymes, such as nucleases, and other substances that exist in intra-cellular and extra-cellular environments.
  • the modification increases nuclease resistance of the guide RNA relative to a guide RNA without the modification, thus it enhances the guide RNA stability.
  • the stability-altering modification is a stability-enhancing modification.
  • the stability-enhancing modification comprises a 2′-O-methyl or a 2′-O—C 1-3 alkyl nucleotide.
  • the stability-enhancing modification comprises a 4′-thioribosyl sugar moiety. In certain embodiments, the stability-enhancing modification comprises a 3′-phosphorothioate group. In certain embodiments, the stability-enhancing modification comprises a 3′-phosphonoacetate group. In certain embodiments, the stability-enhancing modification comprises a nucleotide containing a 3′-thiophosphonoacetate group. In certain embodiments, the stability-enhancing modification comprises a nucleotide containing a 3′-methylphosphonate group. In certain embodiments, the stability-enhancing modification comprises a nucleotide containing a 3′-boranophosphate group. In certain embodiments, the stability-enhancing modification comprises a nucleotide containing a 3′-phosphorodithioate group. In certain embodiments, the stability-enhancing modification comprises an unlocked nucleic acid (“ULNA”) nucleotide.
  • ULNA unlocked nucleic acid
  • the stability-enhancing modification comprises a 2′-O-methyl and a 3′-phosphorothioate group on the same nucleotide. In certain embodiments, the stability-enhancing modification comprises a 2′-O-methyl and a 3′-phosphonoacetate group on the same nucleotide. In certain embodiments, the stability-enhancing modification comprises a 2′-O-methyl and a 3′-thiophosphonoacetate group on the same nucleotide. In certain embodiments, the stability-enhancing modification comprises a 2′-fluoro and a 3′-phosphorothioate group on the same nucleotide.
  • the modification is a specificity-altering modification.
  • specificity enhancement may be achieved by enhancing on-target binding and/or cleavage, or reducing off-target binding and/or cleavage, or a combination of both.
  • specificity reduction may be achieved, for example, by reducing on-target binding and/or cleavage, or increasing off-target binding and/or cleavage, or a combination of both.
  • the specificity-altering modification comprises 5-propynyluracil. In certain embodiments, the specificity-altering modification comprises 5-ethynylcytosine. In certain embodiments, the specificity-altering modification comprises 5-ethynyluracil. In certain embodiments, the specificity-altering modification comprises 5-allylU. In certain embodiments, the specificity-altering modification comprises 5-allylC. In certain embodiments, the specificity-altering modification comprises 5-aminoallylU. In certain embodiments, the specificity-altering modification comprises 5-aminoallylC. In certain embodiments, the specificity-altering modification comprises an abasic nucleotide. In certain embodiments, the specificity-altering modification comprises a Z base.
  • the specificity-altering modification comprises P base. In certain embodiments, the specificity-altering modification comprises a UNA base. In certain embodiments, the specificity-altering modification comprises isoC. In certain embodiments, the specificity-altering modification comprises isoG. In certain embodiments, the specificity-altering modification comprises 5-methyl-pyrimidine. In certain embodiments, the specificity-altering modification comprises x(A,G,C,T,U). In certain embodiments, the specificity-altering modification comprises y(A,G,C,T,U).
  • a gRNA comprises a guide sequence capable of hybridizing to a target polynucleotide, and the guide sequence comprises one or more modifications that alter base pairing of the guide sequence with the target polynucleotide by altering the melting temperature (T m ) of the gRNA:target polynucleotide duplex relative to a similar duplex without the modification.
  • T m melting temperature
  • the modification lowers the T m of the gRNA:target polynucleotide duplex relative to a similar duplex without the modification.
  • the specificity-altering modification lowers the T m of a base pairing interaction.
  • the specificity-enhancing modification lowers the Tm of a first DNA/RNA duplex comprising the guide RNA and target polynucleotide by at least about 1° C., alternatively at least about 2° C., at least about 3° C., at least about 4° C., at least about 5° C., and/or up to about 6° C., alternatively up to about 8° C., alternatively up to about 10° C., alternatively up to about 13° C., for example by lowering the Tm from about 1° C. to about 13° C., alternatively from about 1° C. to about 6° C.
  • the synthetic guide RNA comprises a chemical modification that promotes efficient and titratable transfectability into cells, especially into the nuclei of eukaryotic cells, and reduces immunostimulatory properties in transfected cells.
  • the synthetic guide RNA comprises a chemical modification that promotes effective delivery into and maintaining in an intended cell, tissue, bodily fluid or organism for a duration sufficient to allow the desired gRNA functionality.
  • the synthetic guide RNA comprises a chemical modification that alters the immunostimulatory effect of the guide RNA relative to a guide RNA without the modification.
  • a guide RNA having a chemical modification of the present application has an ON:OFF ratio of greater than 1. In certain embodiments, a guide RNA having a chemical modification of the present application has an ON:OFF ratio of at least about 1.1:1. Thus, in certain embodiments, a guide RNA having a chemical modification of the present application has an ON:OFF ratio of at least about 1.1:1, at least about 0.1.5:1, at least about 2:1, at least about 3:1, at least about 4:1, at least about 5:1, at least about 6:1, at least about 7:1, at least about 8:1, at least about 9:1, at least about 10:1, at least about 15:1, at least about 20:1, at least about 25:1, at least about 30:1, at least about 35:1, at least about 40:1, at least about 45:1, at least about 50:1, at least about 60:1, at least about 70:1, at least about 80:1, at least about 90:1, at least about 95:1, or at least about 99:1.
  • a guide RNA having a chemical modification of the present application has an ON:OFF ratio of at least about 1.5:1 to about 99.9:1. In certain embodiments, a guide RNA having a chemical modification of the present application has an ON:OFF ratio of at least about 10:1 to about 99.9:1.
  • the present technology provides a guide RNA having a combination of two or more modifications.
  • the two modifications are on the same nucleotide (for example, one nucleotide comprises a 2′-O-methyl and a 3′-phosphonoacetate moiety).
  • the two modifications are on two different nucleotides (for example, one nucleotide has a 2′-O-methyl group and another has a 3′-phosphonoacetate moiety).
  • each modification in the guide RNA is the same. In certain embodiments, at least one modification in the guide RNA is different from at least one other modification in the guide RNA. In certain embodiments, the guide RNA comprises a combination of different types of modifications, and at least one type in the combination exists in multiple places in the guide RNA. In certain embodiments, at least one type in the combination appears 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 times in the guide RNA.
  • the modified gRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 modified sugars.
  • the modified gRNA comprises at least 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130 or 140 modified sugars. In certain embodiments, all sugars in a gRNA are modified.
  • the modified gRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 modified internucleotide linkages.
  • the modified gRNA comprises at least 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130 or 140 modified internucleotide linkages.
  • all internucleotide linkages in a gRNA are modified.
  • At least one of the modifications in the combination comprises a 2′-O-methyl, a 2′-fluoro, a 2′-amino, a 2′-deoxy, a 2′-arabino, a 2′-F-arabino, a 2-thiouracil, a 2-aminoadenine, a 5-methylcytosine, a 5-aminoallyluracil, a Z base, a 3′-phosphorothioate, a 3′-phosphonoacetate, a 3′-phosphonoacetate ester, a 3′-thiophosphonoacetate, a 3′-thiophosphonoacetate ester, a 3′-methylphosphonate, a 3′-boranophosphonate, a 3′-phosphorodithioate, or combinations thereof.
  • At least one of the modifications in the combination is an “end” modification such as terminal phosphate, a PEG, a terminal amine, a terminal linker such as a hydrocarbon linker, a substituted hydrocarbon linker, a squarate linker, a triazolo linker, an internal linker such as 2-(4-butylamidofluorescein)propane-1,3-diol bis(phosphodiester) linker, a linker conjugated to a dye, a linker conjugated to a non-fluorescent label, a linker conjugated to a tag or a linker conjugated to a solid support such as for example a bead or microarray.
  • end modification such as terminal phosphate, a PEG, a terminal amine, a terminal linker such as a hydrocarbon linker, a substituted hydrocarbon linker, a squarate linker, a triazolo linker, an internal linker such as 2-(4-butylamid
  • At least two of the modifications in the combination comprise a 2′-O-methyl nucleotide and phosphorothioate internucleotide linkage, a 2′-O-methyl nucleotide and phosphonoacetate internucleotide linkage, or a 2′-O-methyl nucleotide and thiophosphonoacetate internucleotide linkage.
  • the modifications in the combination further comprise a 2-thiouracil, 2-thiocytosine, 4-thiouracil, 6-thioguanine, 2-aminoadenine, 2-aminopurine, pseudouracil, inosine, 7-deazaguanine, 7-deaza-8-azaguanine, 7-deazaadenine, 7-deaza-8-azaadenine, 5-methylcytosine, 5-methyluracil, 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5,6-dehydrouracil, 5-propynylcytosine, 5-propynyluracil, 5-ethynylcytosine, 5-ethynyluracil, 5-allyluracil, 5-allylcytosine, 5-aminoallyl-uracil, 5-aminoallyl-cytosine, or an abasic nucleotide.
  • At least one of the modifications in the combination comprises a 2′-halo-3′-thiophosphonoacetate. In certain embodiments, at least one of the modifications in the combination comprises a 2′-fluoro-3′-phosphorothioate. In certain embodiments, at least one of the modifications in the combination comprises a 2′-fluoro-3′-phosphonoacetate. In certain embodiments, at least one of the modifications in the combination comprises a 2′-fluoro-3′-thiophosphonoacetate. Possible combinations of at least two or three modifications are represented in FIG. 6 and FIG. 7 respectively and are incorporated herein by reference.
  • the guide RNA comprises an oligonucleotide represented by Formula (III) or Formula (IV): W—Y-Q (III); or Y—W—X-Q (IV)
  • Q and W each independently represent a nucleotide or a stretch of nucleotides of the oligonucleotide comprising at least one specificity-enhancing modification and Y and X each independently represent an unmodified portion of the oligonucleotide.
  • W is within the 5′ portion of the guide RNA. In certain embodiments, W is at least partially within the first five (5) nucleotides of the 5′ portion of the guide RNA. In certain embodiments, W is at least partially within the first three (3) nucleotides of the 5′ portion of the guide RNA. In certain embodiments, W is within the internal region (i.e., between the 5′ end and the 3′ end) of the guide RNA.
  • W is at least partially within nucleotides 4 to 20, alternatively within nucleotides 5 to 20 of the guide sequence, alternatively within nucleotides 10 to 20 of the guide sequence, alternatively within nucleotides 13 to 20 of the guide sequence, alternatively within nucleotides 13-14 or 16-19 of the guide sequence, alternatively within nucleotides 13-14 or 16-18 of the guide sequence.
  • At least one of the modifications in the combination enhances stability and specificity of the guide RNA relative to a guide RNA without the modification. In certain embodiments, at least one of the modifications in the combination enhances stability and transfection efficiency of the guide RNA relative to a guide RNA without the modification. In certain embodiments, at least one of the modifications in the combination enhances specificity and transfection efficiency of the guide RNA relative to a guide RNA without the modification.
  • At least one of the modifications in the combination alters the secondary structure of the guide RNA.
  • This modification alters the base-pairing of any of the RNA/RNA internal duplexes in the guide RNA.
  • Some of these modifications increase the base pairing of the RNA/RNA structure or alternatively increase the Tm of the RNA/RNA duplex, whereas other modifications decrease the base pairing (or Tm) of the RNA/RNA duplex or duplexes.
  • Such modifications include base modified nucleotides, particularly UNA nucleotides such as the 2-thiouridine and 2-aminoadenosine pair, the Z/P nucleotide pair, the isoC/isoG pair, the 6-thioG/5-methylpyrimidine pair, and nucleotides with modifications on the sugar or the internucleotide linkages as discussed before.
  • the combination includes at least one modification or a set of modifications that increases nucleases resistance (i.e., stability) with at least one modification or a set of modifications that increases specificity (i.e., reduces off-target effects).
  • the combination includes at least one modification or a set of modifications that increases nucleases resistance (i.e., stability) with at least one modification or a set of modifications that raises the Tm of some bases pairing in the guide RNA.
  • the combination includes at least one modification or a set of modifications that increases nucleases resistance (i.e., stability) with at least one modification or a set of modifications that lowers the Tm of some bases pairing of the guide RNA.
  • the combination includes at least one modification or a set of modifications that increases nuclease resistance (i.e., stability), at least one modification or a set of modifications that increases the Tm of some bases paring in the guide RNA, and at least one modification or a set of modifications that decreases the Tm of some base paring elsewhere in the guide RNA.
  • the combination includes at least one modification or a set of modifications that increases nuclease resistance (i.e., stability) and at least one modification or a set of modifications that increases the binding of the guide RNA to Cas protein.
  • the combination includes at least one modification or a set of modifications that increases nuclease resistance (i.e., stability) and at least one modification or a set of modifications that decreases the binding of the guide RNA to Cas protein.
  • the guide RNA comprises a combination of the different types of modifications.
  • the guide RNA is able to form a complex with a CRISPR-associated-protein.
  • the CRISPR-associated protein is provided by or is derived from a CRISPR-Cas type 1 system, which has an RNA-guided polynucleotide binding and/or nuclease activity.
  • the CRISPR-associated protein is Cas9, a Cas9 mutant, or a Cas9 variant.
  • the CRISPR-associated protein is the Cas9 nuclease from Streptococcus pyogenes .
  • the CRISPR-associated protein is the Cas9 nuclease from Streptococcus thermophilus .
  • the CRISPR-associated protein is the Cas9 nuclease from Staphylococcus aureus .
  • the synthetic guide RNA or a synthetic guide RNA:CRISPR-associated protein complex maintains functionality of natural guide RNA or a complex that does not have modified nucleotides.
  • the functionality includes binding a target polynucleotide.
  • the functionality includes nicking a target polynucleotide.
  • the functionality includes cleaving a target polynucleotide.
  • the target polynucleotide is within a nucleic acid in vitro.
  • the target polynucleotide is within the genome of a cell in vivo or in vitro (such as in cultured cells or cells isolated from an organism). In certain embodiments, the target polynucleotide is a protospacer in DNA.
  • the crRNA segment comprises from 25 to 80 nucleotides. In certain embodiments, the crRNA segment comprises a guide sequence that is capable of hybridizing to a target sequence. In certain embodiments, the guide sequence is complementary to the target sequence or a portion thereof. In certain embodiments, the guide sequence comprises from 15 to 30 nucleotides. In certain embodiments, the crRNA segment comprises a stem sequence. In certain embodiments, the stem sequence comprises from 10 to 50 nucleotides. In certain embodiments, the crRNA segment comprises a 5′-overhang sequence. In certain embodiments, the 5′-overhang sequence comprises from 1 to 10 nucleotides, alternatively 1 to 5 nucleotides, alternatively 1, 2 or 3 nucleotides.
  • the crRNA comprises both (i) a guide sequence that is capable of hybridizing to a target sequence and (ii) a stem sequence.
  • the crRNA comprises (i) a 5′-overhang sequence, (ii) a guide sequence that is capable of hybridizing to a target sequence, and (iii) a stem sequence.
  • the tracrRNA segment comprises a nucleotide sequence that is partially or completely complementary to the stem sequence of the crRNA segment.
  • the tracrRNA segment comprises at least one more duplex structure.
  • the guide RNA is a single-guide RNA, wherein the crRNA segment and the tracrRNA segment are linked through a loop L.
  • the loop L comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.
  • the loop L comprises a nucleotide sequence of GNRA, wherein N represents A, C, G, or U and R represents A or G.
  • the loop L comprises a nucleotide sequence of GAAA.
  • the guide RNA comprises more than one loop.
  • the guide RNA comprises a 5′ portion (i.e., the 5′ half) and a 3′ portion (i.e., the 3′ half).
  • the crRNA segment is 5′ (i.e., upstream) of the tracrRNA segment.
  • the tracrRNA segment is 5′ relative to the crRNA segment.
  • the guide RNA comprises at least two separate RNA strands, for example, a crRNA strand and a separate tracrRNA strand. See, for example, FIG. 2A .
  • each of the strands is a synthetic strand comprising one or more modifications.
  • at least one of the strands is a synthetic strand comprising one or more modifications.
  • the strands function together to guide binding, nicking, or cleaving of a target polynucleotide by a Cas protein, such as Cas9.
  • the crRNA sequence and the tracrRNA sequence are on separate stands and hybridize to each other via two complementary sequences to form a stem or duplex.
  • the guide RNA is a single-guide RNA comprising a crRNA sequence and a tracrRNA sequence. See, for example, FIG. 2B .
  • the crRNA sequence and the tracrRNA sequence are connected by a loop sequence or “loop.”
  • a single-guide RNA comprises a 5′ portion and a 3′ portion, wherein the crRNA sequence is upstream of the tracrRNA sequence.
  • the total length of the two RNA pieces can be about 50-220 (e.g., about 55-200, 60-190, 60-180, 60-170, 60-160, 60-150, 60-140, 60-130, and 60-120) nucleotides in length, such as about 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or 220 nucleotides in length.
  • the single-guide RNA e.g., FIG.
  • 2B can be about 50-220 (e.g., about 55-200, 60-190, 60-180, 60-170, 60-160, 60-150, 60-140, 60-130, and 60-120) nucleotides in length, such as about 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or 220 nucleotides in length.
  • 50-220 e.g., about 55-200, 60-190, 60-180, 60-170, 60-160, 60-150, 60-140, 60-130, and 60-120 nucleotides in length, such as about 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or 220 nucleotides in length.
  • the synthetic guide RNA comprises (i) a crRNA sequence that comprises (a) a guide sequence (e.g., segment G 1 -G n , where each G represents a nucleotide in the guide sequence) capable of hybridizing to a target sequence in a nucleic acid, (b) a first stem sequence (e.g., segment X 1 -X n , where each X represents a nucleotide in the first stem sequence) capable of hybridizing partially or completely to a second stem sequence, and, optionally (c) a 5′-overhang sequence (e.g., segment O 1 -O n , where each O represents a nucleotide in the overhang sequence), and (ii) a tracrRNA sequence that comprises the second stem sequence (e.g., segment Y 1 -Y n , where each Y represents a nucleotide in the second stem sequence).
  • a guide sequence e.g., segment G 1 -G n , where each
  • the tracrRNA sequence further comprises segment T 1 -T n , where each T represents a nucleotide in the tracrRNA sequence.
  • the synthetic guide RNA shown in FIG. 2A includes one or more modifications.
  • the synthetic guide RNA shown in FIG. 2B includes one or more modifications.
  • the modification is located at any point along the length of the crRNA, the tracrRNA, or the single-guide RNA comprising a crRNA segment, a tracrRNA segment, and, optionally, a loop.
  • any nucleotide represented by O, G, X, Y, or T in the synthetic guide RNA shown in FIGS. 2A and 2B may be a modified nucleotide.
  • the guide RNA shown in FIG. 2B represents a single-guide RNA (sgRNA) where the crRNA segment and the tracrRNA segment are connected by a loop having the sequence GNRA, wherein N represents A, C, G, or U, and R represents A or G.
  • the crRNA segment of the guide RNA is 25-70 (e.g., 30-60, 35-50, or 40-45) nucleotides in length.
  • the guide sequence is 12-30 (e.g., 16-25, 17-20, or 15-18) nucleotides in length.
  • a 5′ portion of the crRNA does not hybridize or only partially hybridizes with the target sequence. For example, there can be a 5′-overhang on the crRNA segment.
  • the single-guide RNA comprises a central portion including the stem sequence of the crRNA segment, the stem sequence of the tracrRNA segment, and, optionally, a loop that covalently connects the crRNA segment to the tracrRNA segment.
  • the central segment of the single-guide RNA is 8-60 (e.g., 10-55, 10-50, or 20-40) nucleotides in length.
  • guide RNAs including single-guide RNAs are produced by chemical synthesis using the art of synthetic organic chemistry.
  • the synthetic guide RNAs described herein can be chemically synthesized using methods well-known in the art (such as TBDMS chemistry, TOM Chemistry, ACE chemistry, etc.).
  • the synthetic guide RNAs can be synthesized using TC chemistry by the method described in Dellinger et al. (2011) J. Am. Chem. Soc. 133, 11540; U.S. Pat. No. 8,202,983; and US Patent Application 2010/0076183A1, the contents of which are incorporated by reference in their entireties.
  • TC chemistry refers to the composition and methods of using RNA monomeric nucleotide precursors protected on the 2′-hydroxyl moiety by a thionocarbamate protecting group, to synthesize unmodified RNA or modified RNA comprising one or more modified nucleotides.
  • the ability to chemically synthesize relatively long RNAs (as long as 200-mers or more) using TC-RNA chemistry allows one to produce guide RNAs with special features capable of outperforming those enabled by the four predominant ribonucleotides (A, C, G and U).
  • Some synthetic guide RNAs described herein can also be made using methods known in the art that include in vitro transcription and cell-based expression. For example, 2′-fluoro NTPs can be incorporated into synthetic guide RNAs produced by cell-based expression.
  • Synthesis of guide RNAs can also be accomplished by chemical or enzymatic synthesis of RNA sequences that are subsequently ligated together by enzymes, or chemically ligated by chemical ligation, including but not limited to cyanogen bromide chemistry, “click” chemistry as published by R. Kumar et al. (2007) J. Am. Chem. Soc. 129, 6859-64, or squarate conjugation chemistry as described by K. Hill in WO2013176844 titled “Compositions and methods for conjugating oligonucleotides.”
  • methods are provided for preparing a synthetic guide RNA.
  • the methods comprise selecting a target polynucleotide in a genome; identifying one or more off-target polynucleotide in the genome; identifying one or more shared nucleotide residues, wherein the shared nucleotide residues are present in both the target polynucleotide and the off-target polynucleotide; and designing a synthetic guide RNA, wherein the guide sequence includes a specificity-enhancing modification.
  • the methods can comprise synthesizing the designed guide RNA.
  • the off-target polynucleotide is identified by an algorithm to predict off-target sites as well as their severity such as those found at http://www.rgenome.net/Cas-OFFinder; https://cm.jefferson.edu/Off-Spotter; or http://crispr.mit.edu, or other technique for identifying and quantifying the activation of off-target sites in actual cases, as disclosed in Tsai et al. (2015) Nat. Biotechnol. 33, 187-97; Ran et al. (2015) Nature 520, 186-91; Frock et al. (2015) Nat. Biotechnol. 33, 179-86.
  • the method further comprises identifying at least one distinguishing position between the sequences of the target polynucleotide and the off-target polynucleotide, wherein the target polynucleotide and the off-target polynucleotide have a different nucleotide residue at the at least one distinguishing position, and including in the synthetic guide RNA a nucleotide matching (i.e., complementary to) the nucleotide at the at least one distinguishing position in the target polynucleotide.
  • the present invention provides a set or library of multiple guide RNAs.
  • the library contains two or more guide RNAs disclosed herein.
  • the library can contain from about 10 to about 10′ individual members, e.g., about 10 to about 10 2 , about 10 2 to about 10 3 , about 10 3 to about 10 5 , from about 10 5 to about 10 7 members.
  • An individual member of the library differs from other members of the library at least in the guide sequence, i.e., the DNA targeting segment of the gRNA.
  • each individual member of a library can contain the same or substantially the same nucleotide sequence for the tracrRNA segment as all the other members of the library. In this way, the library can comprise members that target different polynucleotides or different sequences in one or more polynucleotides.
  • the library comprises a collection of guide RNAs having the same sequence and the same modifications in a progressively shifted window that moves across the sequence of the members in the library.
  • the windows collectively cover the entire length of the RNA.
  • the library allows one to conduct high-throughput, multi-target genomic manipulations and analyses.
  • only the DNA-targeting segments of the guide RNAs are varied, while the Cas protein-binding segment is the same.
  • a first portion of the library comprises guide RNAs possessing a Cas-binding segment that recognizes, binds and directs a particular Cas protein and a second portion of the library comprises a different Cas-binding segment that recognizes, binds and directs a different Cas protein (e.g., a Cas protein from a different species), thereby allowing the library to function with two or more orthogonal Cas proteins.
  • induced expression of a first orthogonal Cas protein utilizes the portion of the library which interacts with the first orthogonal Cas protein.
  • induced expression of a first and second orthogonal Cas protein utilizes the portions of the library which interact with the first and second orthogonal Cas proteins, respectively.
  • induced expression of the first and second orthogonal Cas proteins occur at different times. Accordingly, one can carry out large-scale gene editing or gene regulation by specifically manipulating or modifying multiple targets as specified in the library.
  • the library is an “arrayed” library, namely a collection of different features or pools of features in an addressable arrangement. For example, features of an array can be selectively cleaved and transferred to a microtiter plate such that each well in the plate contains a known feature or a known pool of features.
  • the library is synthesized in a 48-well or in a 96-well microtiter plate format or in a 384-well plate.
  • synthesis of the guide RNA of this invention may be conducted on a solid support having a surface to which chemical entities may bind.
  • guide RNAs being synthesized are attached, directly or indirectly, to the same solid support and may form part of an array.
  • An “array” is a collection of separate molecules of known monomeric sequence each arranged in a spatially defined and a physically addressable manner, such that the location of each sequence is known.
  • An “array,” or “microarray’ used interchangeably herein includes any one-dimensional, two-dimensional or substantially two-dimensional (as well as a three-dimensional) arrangement of addressable regions bearing a particular chemical moiety or moieties (such as ligands, e.g., biopolymers such as polynucleotide or oligonucleotide sequences (nucleic acids), polypeptides (e.g., proteins), carbohydrates, lipids, etc.) associated with that region.
  • ligands e.g., biopolymers such as polynucleotide or oligonucleotide sequences (nucleic acids), polypeptides (e.g., proteins), carbohydrates, lipids, etc.
  • An array is “addressable” when it has multiple regions of different moieties (e.g., different polynucleotide sequences) such that a region (i.e., a “feature” of the array) at a particular predetermined location (i.e., an “address”) on the array will detect a particular target or class of targets (although a feature may incidentally detect non-targets of that feature).
  • Array features are typically, but need not be, separated by intervening spaces. The number of features that can be contained on an array will largely be determined by the surface area of the substrate, the size of a feature and the spacing between features. Arrays can have densities of up to several hundred thousand or more features per cm 2 , such as 2,500 to 200,000 features/cm 2 . The features may or may not be covalently bonded to the substrate.
  • Suitable solid supports may have a variety of forms and compositions and derive from naturally occurring materials, naturally occurring materials that have been synthetically modified, or synthetic materials.
  • suitable support materials include, but are not limited to, silicas, silicon and silicon oxides, teflons, glasses, polysaccharides such as agarose (e.g., Sepharose® from Pharmacia) and dextran (e.g., Sephadex® and Sephacyl®, also from Pharmacia), polyacrylamides, polystyrenes, polyvinyl alcohols, copolymers of hydroxyethyl methacrylate and methyl methacrylate, and the like.
  • the solid support is a plurality of beads.
  • the present invention can be used to prepare arrays of guide RNAs wherein the oligonucleotides are either synthesized on the array, or attached to the array substrate post-synthesis. Subsequently, the guide RNAs or a pool or a plurality of pools of guide RNAs can optionally and selectively be cleaved from the array substrate and be used as a library or libraries.
  • the present invention also provides various crRNAs that comprise the chemical modification(s) as described for guide RNAs, as described herein.
  • the crRNAs can function in a multi-segment guide RNA, such as a dual guide.
  • the present invention provides a synthetic crRNA comprising (i) a guide sequence capable of hybridizing to a target polynucleotide, the target polynucleotide comprising a target sequence adjacent to a PAM site, and (ii) a stem sequence; wherein the guide sequence consists of 20-N nucleotides, where N is an integer between ⁇ 10 and 10 (optionally between ⁇ 10 and 6); wherein the guide sequence comprises at least one modification, and the crRNA results in higher specificity for the target polynucleotide or higher gRNA functionality than a corresponding crRNA without the modification.
  • modifications of interest are described elsewhere in this disclosure, including but not limited to at least one modification at nucleotides 4-N to 20-N, or at at least one nucleotide selected from 4-N, 5-N, 7-N, 9-N, 10-N and 11-N, of the guide sequence.
  • Various embodiments of the particular modification(s) and target polynucleotides are also described herein.
  • a functional CRISPR-Cas system also requires a protein component (e.g., a Cas protein, which may be a Cas nuclease) that provides a desired activity, such as target binding or target nicking/cleaving.
  • a desired activity such as target binding or target nicking/cleaving.
  • the desired activity is target binding.
  • the desired activity is target nicking or target cleaving.
  • the desired activity also includes a function provided by a polypeptide that is covalently fused to a Cas protein, as disclosed herein.
  • the desired activity also includes a function provided by a polypeptide that is covalently fused to a nuclease-deficient Cas protein, as disclosed herein.
  • the Cas protein can be introduced into an in vitro or in vivo system as a purified or non-purified (i) Cas protein or (ii) mRNA encoded for expression of the Cas protein or (iii) linear or circular DNA encoded for expression of the protein. Any of these 3 methods of providing the Cas protein are well known in the art and are implied interchangeably when mention is made herein of a Cas protein or use of a Cas protein.
  • the Cas protein is constitutively expressed from mRNA or DNA.
  • the expression of Cas protein from mRNA or DNA is inducible or induced.
  • the Cas protein is chemically synthesized (see e.g., Creighton, “Proteins: Structures and Molecular Principles,” W.H. Freeman & Co., NY, 1983), or produced by recombinant DNA technology as described herein.
  • skilled artisans may consult Frederick M. Ausubel et al., “Current Protocols in Molecular Biology,” John Wiley & Sons, 2003; and Sambrook et al., “Molecular Cloning, A Laboratory Manual,” Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 2001).
  • the Cas protein is provided in purified or isolated form. In certain embodiments, the Cas protein is provided at about 80%, about 90%, about 95%, or about 99% purity. In certain embodiments, the Cas protein is provided as part of a composition. In certain embodiments, the Cas protein is provided in aqueous compositions suitable for use as, or inclusion in, a composition for an RNA-guided nuclease reaction. Those of skill in the art are well aware of the various substances that can be included in such nuclease reaction compositions.
  • a Cas protein is provided as a recombinant polypeptide.
  • the recombinant polypeptide is prepared as a fusion protein.
  • a nucleic acid encoding the Cas protein is linked to another nucleic acid encoding a fusion partner, e.g., glutathione-S-transferase (GST), 6 ⁇ -His epitope tag, or M13 Gene 3 protein.
  • GST glutathione-S-transferase
  • 6 ⁇ -His epitope tag 6 ⁇ -His epitope tag
  • M13 Gene 3 protein e.g., M13 Gene 3 protein.
  • Suitable host cells can be used to expresses the fusion protein.
  • the fusion protein is isolated by methods known in the art.
  • the fusion protein can be further treated, e.g., by enzymatic digestion, to remove the fusion partner and obtain the Cas protein.
  • Cas protein:gRNA complexes can be made with recombinant technology using a host cell system or an in vitro translation-transcription system known in the art. Details of such systems and technology can be found in e.g., WO2014144761 WO2014144592, WO2013176772, US20140273226, and US20140273233, the contents of which are incorporated herein by reference in their entireties.
  • a Cas protein comprises a protein derived from a CRISPR-Cas type I, type II, or type III system, which has an RNA-guided polynucleotide binding and/or nuclease activity.
  • suitable Cas proteins include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, C
  • the Cas protein is derived from a type II CRISPR-Cas system. In certain embodiments, the Cas protein is or is derived from a Cas9 protein. In certain embodiments, the Cas protein is or is derived from a bacterial Cas9 protein, including those identified in WO2014144761. In certain embodiments, the Cas protein is or is derived from a Streptococcus sp. or Staphylococcus sp. Cas9 protein. In certain embodiments, the Cas protein is or is derived from the Streptococcus thermophilus Cas9 protein. In certain embodiments, the Cas protein is or is derived from the Streptococcus pyogenes Cas9 protein. In certain embodiments, the Cas protein is or is derived from the Staphylococcus aureus Cas9 protein. In certain embodiments, the Cas protein is or is derived from the Streptococcus thermophilus Cas9 protein.
  • the wild type Cas protein is a Cas9 protein. In certain embodiments, the wild type Cas9 protein is the Cas9 protein from S. pyogenes (SEQ ID NO: 115). In certain embodiments, the protein or polypeptide can comprise, consist of, or consist essentially of a fragment of SEQ ID NO: 115.
  • a Cas protein includes at least one RNA binding domain, which interacts with the guide RNA.
  • the Cas protein is modified to increase nucleic acid binding affinity and/or specificity, alter an enzymatic activity, and/or change another property of the protein.
  • nuclease i.e., DNase, RNase
  • the Cas protein can be truncated to remove domains that are not essential for the function of the protein.
  • the Cas protein is truncated or modified to optimize the activity of the effector domain.
  • the Cas protein includes a nuclear localization sequence (NLS) that effects importation of the NLS-tagged Cas protein into the nucleus of a living cell.
  • the Cas protein includes two or more modifications.
  • the Cas protein can be a mutant of a wild type Cas protein (such as Cas9) or a fragment thereof.
  • the Cas protein can be derived from a mutant Cas protein.
  • the amino acid sequence of the Cas9 protein can be modified to alter one or more properties (e.g., nuclease activity, binding affinity, stability to proteases, etc.) of the protein.
  • domains of the Cas9 protein not involved in RNA-guided cleavage can be eliminated from the protein such that the modified Cas9 protein is smaller than the wild type Cas9 protein.
  • the present system utilizes the Cas9 protein from S. pyogenes , either as encoded in bacteria or codon-optimized for expression in eukaryotic cells. Shown below is the amino acid sequence of wild type S. pyogenes Cas9 protein sequence (SEQ ID NO: 115, available at www.uniprot.org/uniprot/Q99ZW2).
  • a Cas9 protein generally has at least two nuclease (e.g., DNase) domains.
  • a Cas9 protein can have a RuvC-like nuclease domain and an HNH-like nuclease domain.
  • the RuvC and HNH domains work together to cut both strands in a target site to make a double-stranded break in the target polynucleotide.
  • a mutant Cas9 protein is modified to contain only one functional nuclease domain (either a RuvC-like or an HNH-like nuclease domain).
  • the mutant Cas9 protein is modified such that one of the nuclease domains is deleted or mutated such that it is no longer functional (i.e., the nuclease activity is absent).
  • the mutant is able to introduce a nick into a double-stranded polynucleotide (such protein is termed a “nickase”) but not able to cleave the double-stranded polynucleotide.
  • nickase such protein is termed a “nickase”
  • an aspartate to alanine (D10A) conversion in a RuvC-like domain converts the Cas9-derived protein into a nickase.
  • H840A histidine to alanine
  • N863A arsparagine to alanine
  • both the RuvC-like nuclease domain and the HNH-like nuclease domain are modified or eliminated such that the mutant Cas9 protein is unable to nick or cleave the target polynucleotide.
  • all nuclease domains of the Cas9-derived protein are modified or eliminated such that the Cas9-derived protein lacks all nuclease activity.
  • a Cas9 protein that lacks some or all nuclease activity relative to a wild-type counterpart nevertheless, maintains target recognition activity to a greater or lesser extent.
  • any or all of the nuclease domains can be inactivated by one or more deletion mutations, insertion mutations, and/or substitution mutations using well-known methods, such as site-directed mutagenesis, PCR-mediated mutagenesis, and total gene synthesis, as well as other methods known in the art.
  • the “Cas mutant” or “Cas variant” is at least 50% (e.g., any number between 50% and 100%, inclusive, e.g., 50%, 60%, 70%, 80%, 90%, 95%, 98%, and 99%) identical to SEQ ID NO: 115.
  • the “Cas mutant” or “Cas variant” binds to an RNA molecule (e.g., a sgRNA).
  • the “Cas mutant” or “Cas variant” is targeted to a specific polynucleotide sequence via the RNA molecule.
  • the Cas protein is fused to another protein or polypeptide heterologous to the Cas protein to create a fusion protein.
  • the heterologous sequence includes one or more effector domains, such as a cleavage domain, a transcriptional activation domain, a transcriptional repressor domain, or an epigenetic modification domain. Additional examples of the effector domain include a nuclear localization signal, cell-penetrating or translocation domain, or a marker domain.
  • the effector domain is located at the N-terminal, the C-terminal, or in an internal location of the fusion protein.
  • the Cas protein of the fusion protein is or is derived from a Cas9 protein.
  • the Cas protein of the fusion protein is or is derived from a modified or mutated Cas protein in which all the nuclease domains have been inactivated or deleted. In certain embodiments, the Cas protein of the fusion protein is or is derived from a modified or mutated Cas protein that lacks nuclease activity. In certain embodiments, the RuvC and/or HNH domains of the Cas protein are modified or mutated such that they no longer possess nuclease activity.
  • the effector domain of the fusion protein is a cleavage domain.
  • a “cleavage domain” refers to a domain that cleaves DNA.
  • the cleavage domain can be obtained from any endonuclease or exonuclease.
  • Non-limiting examples of endonucleases from which a cleavage domain can be derived include restriction endonucleases and homing endonucleases. See, for example, New England Biolabs Catalog or Belfort et al. (1997) Nucleic Acids Res. 25, 3379-88.
  • cleave DNA is known (e.g., 51 Nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease; yeast HO endonuclease). See also Linn et al. (eds.) “Nucleases,” Cold Spring Harbor Laboratory Press, 1993. One or more of these enzymes (or functional fragments thereof) can be used as a source of cleavage domains.
  • the cleavage domain can be derived from a type II-S endonuclease.
  • Type II-S endonucleases cleave DNA specifically at sites that are typically several base pairs away from the DNA recognition site of the endonuclease and, as such, have separable recognition and cleavage domains. These enzymes generally are monomers that transiently associate to form dimers to cleave each strand of DNA at staggered locations.
  • suitable type II-S endonucleases include BfiI, BpmI, BsaI, BsgI, BsmBI, BsmI, BspMI, FokI, MbolI, and SapI.
  • the cleavage domain of the fusion protein is a FokI cleavage domain or a fragment or derivative thereof. See Miller et al. (2007) Nat. Biotechnol. 25, 778-85; Szczpek et al. (2007) Nat. Biotechnol. 25, 786-93; Doyon et al. (2011) Nat. Methods, 8, 74-81.
  • the effector domain of the fusion protein is a transcriptional activation domain.
  • a transcriptional activation domain interacts with transcriptional control elements and/or transcriptional regulatory proteins (i.e., transcription factors, RNA polymerases, etc.) to increase and/or activate transcription of a gene.
  • the transcriptional activation domain is a herpes simplex virus VP16 activation domain, VP64 (which is a tetrameric derivative of VP16), a NF ⁇ B p65 activation domain, p53 activation domains 1 and 2, a CREB (cAMP response element binding protein) activation domain, an E2A activation domain, or an NFAT (nuclear factor of activated T-cells) activation domain.
  • the transcriptional activation domain is Gal4, Gcn4, MLL, Rtg3, Gln3, Oaf1, Pip2, Pdr1, Pdr3, Pho4, or Leu3.
  • the transcriptional activation domain may be wild type, or it may be a modified or truncated version of the original transcriptional activation domain.
  • the effector domain of the fusion protein is a transcriptional repressor domain.
  • a transcriptional repressor domain interacts with transcriptional control elements and/or transcriptional regulatory proteins (i.e., transcription factors, RNA polymerases, etc.) to decrease and/or prohibit transcription of a gene.
  • the transcriptional repressor domains is inducible cAMP early repressor (ICER) domains, Kruppel-associated box A (KRAB-A) repressor domains, YY1 glycine rich repressor domains, Sp1-like repressors, E(spI) repressors, I ⁇ B repressor, or MeCP2.
  • the effector domain of the fusion protein is an epigenetic modification domain.
  • epigenetic modification domains alter gene expression by modifying the histone structure and/or chromosomal structure.
  • the epigenetic modification domains is a histone acetyltransferase domain, a histone deacetylase domain, a histone methyltransferase domain, a histone demethylase domain, a DNA methyltransferase domain, or a DNA demethylase domain.
  • the fusion protein further comprises at least one additional domain.
  • suitable additional domains include nuclear localization signals (NLSs), cell-penetrating or translocation domains, and marker domains.
  • NLS generally comprises a stretch of basic amino acids. See, e.g., Lange et al. (2007) J. Biol. Chem. 282, 5101-5.
  • the NLS is a monopartite sequence, such as PKKKRKV (SEQ ID NO: 116) or PKKKRRV (SEQ ID NO: 117).
  • the NLS is a bipartite sequence.
  • the NLS is KRPAATKKAGQAKKKK (SEQ ID NO: 118).
  • the fusion protein comprises at least one cell-penetrating domain.
  • the cell-penetrating domain is a cell-penetrating peptide sequence derived from the HIV-1 TAT protein.
  • the TAT cell-penetrating sequence can be GRKKRRQRRRPPQPKKKRKV (SEQ ID NO: 119).
  • the cell-penetrating domain is TLM (PLSSIFSRIGDPPKKKRKV; SEQ ID NO: 120), a cell-penetrating peptide sequence derived from the human hepatitis B virus.
  • the fusion protein comprises at least one marker domain.
  • marker domains include fluorescent proteins, purification tags, and epitope tags.
  • the marker domain is a fluorescent protein.
  • suitable fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreen1), yellow fluorescent proteins (e.g. YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow1), blue fluorescent proteins (e.g.
  • EBFP EBFP2, Azurite, mKalama1, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g. ECFP, Cerulean, CyPet, AmCyan1, Midoriishi-Cyan), red fluorescent proteins (mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRed1, AsRed2, eqFP611, mRasberry, mStrawberry, Jred), orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato) and any other suitable fluorescent protein.
  • cyan fluorescent proteins e.g. ECFP, Cerulean, CyPet, AmCyan1, Midoriishi-Cy
  • the marker domain is a purification tag and/or an epitope tag.
  • tags include, but are not limited to, glutathione-S-transferase (GST), chitin binding protein (CBP), maltose binding protein, thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, HA, nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6 ⁇ His, biotin carboxyl carrier protein (BCCP), and calmodulin.
  • GST glutathione-S-transferase
  • CBP chitin binding protein
  • TRX thioredoxin
  • poly(NANP) tandem affinity purification
  • TAP tandem affinity purification
  • the present invention provides a method for cleaving a target polynucleotide with a Cas protein.
  • the method comprises contacting the target polynucleotide with (i) a guide RNA or a set of guide RNA molecules described herein, and (ii) a Cas protein.
  • the method results in a double-strand break in the target polynucleotide.
  • the Cas protein is a Cas protein having a single-strand nicking activity.
  • the method results in a single-strand break in the target polynucleotide.
  • a complex comprising a guide RNA and Cas protein having a single-strand nicking activity is used for sequence-targeted single-stranded DNA cleavage, i.e., nicking.
  • the present invention provides a method for cleaving two or more target polynucleotides with a Cas protein.
  • the method comprises contacting the target polynucleotides with (i) a set of guide RNA molecules described herein, and (ii) a Cas protein.
  • the method results in double-strand breaks in the target polynucleotides.
  • the Cas protein is a Cas protein having a single-strand nicking activity.
  • the method results in single-strand breaks in the target polynucleotides.
  • a complex comprising a guide RNA and Cas protein having a single-strand nicking activity is used for sequence-targeted single-stranded DNA cleavage, i.e., nicking.
  • the present invention provides a method for binding a target polynucleotide with a Cas protein.
  • the method comprises contacting the target polynucleotide with (i) a guide RNA or a set of guide RNA molecules described herein and (ii) a Cas protein, to result in binding of the target polynucleotide with the Cas protein.
  • the Cas protein is a Cas variant.
  • the Cas variant lacks some or all nuclease activity relative to a counterpart wild-type Cas protein.
  • the present invention provides a method for binding two or more target polynucleotides with a Cas protein.
  • the method comprises contacting the target polynucleotides with (i) a set of RNA molecules described herein and (ii) a Cas protein, to result in binding of the target polynucleotides with the Cas protein.
  • the Cas protein is a Cas variant.
  • the Cas variant lacks some or all nuclease activity relative to a counterpart wild-type Cas protein.
  • the present invention provides a method for targeting a Cas protein to a target polynucleotide.
  • the method comprises contacting the Cas protein with a guide RNA or a set of guide RNA molecules described herein.
  • the method results in formation of a guide RNA:Cas protein complex.
  • the Cas protein is a wild type Cas9 protein.
  • the Cas protein is a mutant or variant of a Cas9 protein.
  • the Cas protein is a Cas protein having a single-strand nicking activity.
  • the Cas protein is a Cas protein lacking nuclease activity (e.g., a nuclease-deficient mutant of Cas protein).
  • the Cas protein is part of a fusion protein (e.g., a fusion protein comprising (i) the Cas protein and (ii) a heterologous polypeptide).
  • the present invention provides a method for targeting a Cas protein to two or more target polynucleotides.
  • the method comprises contacting the Cas protein with a set of guide RNA molecules described herein.
  • the method results in formation of a guide RNA:Cas protein complex.
  • the Cas protein is a wild type Cas9 protein.
  • the Cas protein is a mutant or variant of a Cas9 protein.
  • the Cas protein is a Cas protein having a single-strand nicking activity.
  • the Cas protein is a Cas protein lacking nuclease activity (e.g., a nuclease-deficient mutant of Cas protein).
  • the Cas protein is part of a fusion protein (e.g., a fusion protein comprising (i) the Cas protein or and (ii) a heterologous polypeptide).
  • the present invention provides a method of selecting a synthetic guide RNA.
  • the method involves “walking” an MP modification across the guide sequence portion of a gRNA to identify which position or positions in the guide sequence enhance specificity due to the location of the MP modification.
  • the magnitude of the specificity enhancement may be assessed for each position tested with the on-target versus off-target cleavage ratio, the cleavage percentage at the target site and the cleavage percentage at one or more off-target sites, amid/or the specificity score, thus determining which modified position or positions alters the specificity of the gRNA and to what extent.
  • the incremental walking of a single MP across the guide sequence may also identify positions for potential synergistic improvements in specificity resulting from one or more combinations of chemical modifications among the positions tested.
  • the method comprises providing at least a first synthetic guide RNA and a second synthetic guide RNA, both comprising the same sequences of (a) a crRNA segment comprising (i) a guide sequence capable of hybridizing to a target polynucleotide, (ii) a stem sequence; and (b) a tracrRNA segment comprising a nucleotide sequence that is partially or completely complementary to the stem sequence, wherein the first synthetic guide RNA comprises an MP modification at a first position of the guide sequence, and the second synthetic guide RNA comprises an MP modification at a second position of the guide sequence; forming a first gRNA:Cas protein complex comprising a Cas protein and the first synthetic guide RNA, contacting the target polynucleotide with the first gRNA:Cas protein complex, and cleaving, nicking or binding the target polynucleotide; forming a second gRNA:Cas protein complex comprising a Cas protein and the second synthetic guide RNA,
  • the first and second gRNA:Cas protein complexes are tested together in a competitive assay, such as by labeling of the first and second gRNAs with different fluorophores.
  • the first and second gRNA:Cas protein complexes are tested individually in equivalent or split samples assayed in parallel or sequentially.
  • the specificity-enhancing modification comprises 2′-O-methyl-3′-phosphonoacetate (MP), 2′-O-methyl-3′-thiophosphonoacetate (MSP), 2′-deoxy-3′-phosphonoacetate (DP), 2′-deoxy-3′-thiophosphonoacetate (DSP), or a combination thereof.
  • the chemical modification comprises a 2′ modification that confers a C3′-endo sugar pucker and a phosphonoacetate or thiophosphonoacetate linkage modification.
  • the 2′ modification is selected from 2′-F and 2′-O-(2-methoxyethyl).
  • the first and second synthetic guide RNAs comprise a specificity-enhancing modification at different nucleotide positions in the guide sequence portions.
  • the specificity is determined based on ON target cleavage activity, OFF target cleavage activity, ON:OFF ratio, specificity score, or a combination thereof.
  • the method comprises providing a first through twentieth synthetic guide RNA comprising a specificity-enhancing modification at different nucleotide positions in the guide sequence portions, forming a gRNA:Cas protein complex using each of the synthetic guide RNAs, contacting the target polynucleotide with the gRNA:Cas protein complex, cleaving, nicking or binding the target polynucleotide and measuring the specificity of each synthetic guide RNA, and identifying one or more modified positions that provide the greatest specificity enhancement.
  • the gRNA further comprises stability-enhancing end modifications.
  • the stability enhancing end modifications comprise 2′-O-methyl-3′-phosphonoacetate (MP), 2′-O-methyl-3′-thiophosphonoacetate (MSP), 2′-O-methyl-3′-phosphorothioate (MS), 2′-deoxy-3′-phosphonoacetate (DP), 2′-deoxy-3′-thiophosphonoacetate (DSP), 2′-fluoro-3′-phosphonoacetate (FP), 2′-fluoro-3′-thiophosphonoacetate (FSP), 2′-fluoro-3′-phosphorothioate (FS), or a combination thereof at the 5′ end and/or the 3′ end of the gRNA.
  • MP 2′-O-methyl-3′-thiophosphonoacetate
  • MS 2′-O-methyl-3′-phosphorothioate
  • DP 2′-deoxy-3′-phosphonoacetate
  • DSP 2′-fluoro-3′-phosphonoacetate
  • FP 2′-
  • the guide RNA is introduced into a cell by transfection.
  • Techniques for RNA transfection are known in the art and include electroporation and lipofection. Effective techniques for RNA transfection depend mostly on cell type. See, e.g., Lujambio et al. (Spanish National Cancer Centre) Cancer Res. February 2007, which describes transfection of HTC-116 colon cancer cells and uses Oligofectamine (Invitrogen) for transfection of commercially obtained, modified miRNA or precursor miRNA. See also, Cho et al. (Seoul National Univ.) Nat. Biotechnol .
  • RNA has been delivered in non-pathogenic E. coli coated with Invasin protein (to facilitate uptake into cells expressing ⁇ -1 integrin protein) and with the E. coli encoded to express lysteriolysin O pore-forming protein to permit the shRNA to pass from the E. coli into the cytoplasm. See also Cho et al. (Seoul National Univ.) Nat. Biotechnol . March 2013.
  • guide RNA or a delivery vehicle containing guide RNA is targeted to a particular tissue or body compartment.
  • synthetic carriers are decorated with cell-specific ligands or aptamers for receptor uptake, e.g., RNA encased in cyclodextrin nanoparticles coated with PEG and functionalized with human transferrin protein for uptake via the transferrin receptor which is highly expressed in tumor cells. Further approaches are described herein below or known in the art.
  • modified guide RNA was introduced into K562 cells, human primary T cells, and CD34+ hematopoietic stem and progenitor cells (HSPCs).
  • the modified guide RNA significantly enhanced genome editing efficiencies in human cells, including human primary T cells and CD34+ HSPCs as compared to unmodified guide RNA.
  • Examples of other uses include genomic editing and gene expression regulation as described below.
  • the present invention provides a method for genomic editing to modify a DNA sequence in vivo or in vitro (“in vitro” includes, without being limited to, a cell-free system, a cell lysate, an isolated component of a cell, and a cell outside of a living organism).
  • the DNA sequence may comprise a chromosomal sequence, an episomal sequence, a plasmid, a mitochondrial DNA sequence, or a functional intergenic sequence, such as an enhancer sequence or a DNA sequence for a non-coding RNA.
  • the method comprises contacting the DNA sequence with (i) a guide RNA or a set of guide RNA molecules described herein, and (ii) a Cas protein.
  • the DNA sequence is contacted outside of a cell.
  • the DNA sequence is located in the genome within a cell and is contacted in vitro or in vivo.
  • the cell is within an organism or tissue.
  • the cell is a human cell, a non-human mammalian cell, a stem cell, a non-mammalian vertebrate cell, an invertebrate cell, a plant cell, a single cell organism, or an embryo.
  • the guide RNA aids in targeting the Cas protein to a targeted site in the DNA sequence.
  • the Cas protein cleaves at least one strand of the DNA sequence at the targeted site.
  • the Cas protein cleaves both strands of the DNA sequence at the targeted site.
  • the method further comprises introducing the Cas protein into a cell or another system.
  • the Cas protein is introduced as a purified or non-purified protein.
  • the Cas protein is introduced via an mRNA encoding the Cas protein.
  • the Cas protein is introduced via a linear or circular DNA encoding the Cas protein.
  • the cell or system comprises a Cas protein or a nucleic acid encoding a Cas protein.
  • a double-stranded break can be repaired via an error-prone, non-homologous end-joining (“NHEJ”) repair process.
  • NHEJ non-homologous end-joining
  • a double-stranded break can be repaired by a homology-directed repair (HDR) process such that a donor sequence in a donor polynucleotide can be integrated into or exchanged with the targeted DNA sequence.
  • HDR homology-directed repair
  • the method further comprises introducing at least one donor polynucleotide into the cell or system.
  • the donor polynucleotide comprises at least one homologous sequence having substantial sequence identity with a sequence on either side of the targeted site in the DNA sequence.
  • the donor polynucleotide comprises a donor sequence that can be integrated into or exchanged with the DNA sequence via homology-directed repair, such as homologous recombination.
  • the donor polynucleotide includes an upstream homologous sequence and a downstream homologous sequence, each of which have substantial sequence identity to sequences located upstream and downstream, respectively, of the targeted site in the DNA sequence.
  • sequence similarities permit, for example, homologous recombination between the donor polynucleotide and the targeted DNA sequence such that the donor sequence can be integrated into (or exchanged with) the DNA sequence targeted.
  • the target site(s) in the DNA sequence spans or is adjacent to a mutation, e.g., point mutation, a translocation or an inversion which may cause or be associated with a disorder.
  • the method comprises correcting the mutation by introducing into the cell or system at least one donor polynucleotide comprising (i) a wild-type counterpart of the mutation and (ii) at least one homologous sequence having substantial sequence identity with a sequence on one side of the targeted site in the DNA sequence.
  • the donor polynucleotide comprises a homologous sequence having substantial sequence identity with a sequence on both sides of the targeted site in the DNA sequence.
  • the donor polynucleotide comprises an exogenous sequence that can be integrated into or exchanged with the targeted DNA sequence via a homology-directed repair process, such as homologous recombination.
  • the exogenous sequence comprises a protein coding gene, which, optionally, is operably linked to an exogenous promoter control sequence.
  • a cell upon integration of the exogenous sequence, a cell can express a protein encoded by the integrated gene.
  • the exogenous sequence is integrated into the targeted DNA sequence such that its expression in the recipient cell or system is regulated by the exogenous promoter control sequence. Integration of an exogenous gene into the targeted DNA sequence is termed a “knock in.”
  • the exogenous sequence can be a transcriptional control sequence, another expression control sequence, an RNA coding sequence, and the like.
  • the donor polynucleotide comprises a sequence that is essentially identical to a portion of the DNA sequence at or near the targeted site, but comprises at least one nucleotide change.
  • the donor sequence comprises a modified or mutated version of the DNA sequence at or near the targeted site such that, upon integration or exchange with the targeted site, the resulting sequence at the targeted site comprises at least one nucleotide change.
  • the at least one nucleotide change is an insertion of one or more nucleotides, a deletion of one or more nucleotides, a substitution of one or more nucleotides, or combinations thereof.
  • the cell may produce a modified gene product from the targeted DNA sequence.
  • the methods are for multiplex applications.
  • the methods comprise introducing a library of guide RNAs into the cell or system.
  • the library comprises at least 10 unique guide sequences.
  • the library comprises at least 100 unique guide sequences.
  • the library comprises at least 1,000 unique guide sequences.
  • the library comprises at least 10,000 unique guide sequences.
  • the library comprises at least 100,000 unique guide sequences.
  • the library comprises at least 1,000,000 unique guide sequences.
  • the library targets at least 10 different polynucleotides or at least 10 different sequences within one or more polynucleotides.
  • the library targets at least 100 different polynucleotides or at least 100 different sequences within one or more polynucleotides. In certain embodiments, the library targets at least 1,000 different polynucleotides or at least 1,000 different sequences within one or more polynucleotides. In certain embodiments, the library targets at least 10,000 different polynucleotides or at least 10,000 different sequences within one or more polynucleotides. In certain embodiments, the library targets at least 100,000 different polynucleotides or at least 100,000 different sequences within one or more polynucleotides. In certain embodiments, the library targets at least 1,000,000 different polynucleotides or at least 1,000,000 different sequences within one or more polynucleotides.
  • Embodiments of the present invention are useful in methods for genomic editing to modify a target polynucleotide, for example a DNA sequence, in a mammalian cell.
  • the DNA sequence is a chromosomal sequence. In certain embodiments, the DNA sequence is a protein-coding sequence. In certain embodiments, the DNA sequence is a functional intergenic sequence, such as an enhancer sequence or a non-coding sequence. In certain embodiments, the DNA is part of a human gene.
  • the human gene is the clathrin light chain (CLTA1) gene, the human interleukin 2 receptor gamma (IL2RG) gene, the human cytotoxic T-lymphocyte-associated protein 4 (CLTA4) gene, the human Vascular Endothelial Growth Factor A gene (VEGFA), or the human hemoglobin beta (HBB) gene which can harbor mutations responsible for sickle cell anemia and thalassemias.
  • CLTA1 clathrin light chain
  • IL2RG human interleukin 2 receptor gamma
  • CLTA4 human cytotoxic T-lymphocyte-associated protein 4
  • VEGFA Vascular Endothelial Growth Factor A gene
  • HBB hemoglobin beta
  • the target polynucleotide is a HBB polynucleotide, a VEGFA polynucleotide, an IL2RG polynucleotide, a CLTA1 polynucleotide, or a CLTA4 polynucleotide.
  • a synthetic guide RNA comprises a guide sequence capable of hybridizing to an HBB, IL2RG, CLTA1, VEGFA, or CLTA4 polynucleotide.
  • the guide sequence consists of nucleotides 1 through 20-N, counted from the 5′ end of the guide sequence, N being an integer between ⁇ 10 and 0, and the guide sequence comprises at least one specificity-enhancing modification at nucleotide 4-N, 5-N, 7-N, 9-N, 10-N, or 11-N.
  • the guide sequence capable of hybridizing to one of the above target polynucleotides has a chemical modification at nucleotide 11-N.
  • the guide sequence capable of hybridizing to one of the above target polynucleotides has a chemical modification at nucleotide 5-N. In certain embodiments, the guide sequence capable of hybridizing to one of the above target polynucleotides has a chemical modification at nucleotide 7-N. In certain embodiments, the guide sequence capable of hybridizing to one of the above target polynucleotides has a chemical modification at nucleotide 10-N. In certain embodiments, the guide sequence capable of hybridizing to one of the above target polynucleotides has a chemical modification at nucleotide 9-N. In certain embodiments, the guide sequence capable of hybridizing to one of the above target polynucleotides has a chemical modification at nucleotide 4-N. In certain embodiments, N equals zero.
  • the guide sequence consists of nucleotides 1 through 19, counted from the 5′ end of the guide sequence, and at least one chemical modification at one of nucleotides 3, 4, 6, 8, 9, or 10. In certain embodiments, the guide sequence consists of nucleotides 1 through 18, counted from the 5′ end of the guide sequence, and at least one chemical modification at one of nucleotides 2, 3, 5, 7, 8, or 9. In certain embodiments, the guide sequence consists of nucleotides 1 through 17, counted from the 5′ end of the guide sequence, and at least one chemical modification at one of nucleotides 1, 2, 4, 6, 7, or 8.
  • the guide sequence consists of nucleotides 1 through 16, counted from the 5′ end of the guide sequence, and at least one chemical modification at one of nucleotides 1, 3, 5, 6, or 7. In certain embodiments, the guide sequence consists of nucleotides 1 through 15, counted from the 5′ end of the guide sequence, and at least one chemical modification at one of nucleotides 2, 4, 5, or 6. In certain embodiments, the guide sequence consists of nucleotides 1 through 14, counted from the 5′ end of the guide sequence, and at least one chemical modification at one of nucleotides 1, 3, 4, or 5.
  • the chemical modification comprises 2′-O-methyl-3′-phosphonoacetate (MP), 2′-O-methyl-3′-thiophosphonoacetate (MSP), 2′-deoxy-3′-phosphonoacetate (DP), 2′-deoxy-3′-thiophosphonoacetate (DSP), or a combination thereof.
  • the chemical modification comprises a 2′-modification that confers a C3′-endo sugar pucker and a phosphonoacetate or thiophosphonoacetate linkage modification.
  • the 2′-modification is selected from 2′-F and 2′-O-(2-methoxyethyl).
  • the mammalian cell is a human cell.
  • the human cell is a primary human cell.
  • the primary human cell is a human primary T cell.
  • the human primary T cell may be stimulated or unstimulated.
  • the human cell is a stem/progenitor cell, such as a CD34+ hematopoietic stem and progenitor cell (HSPC).
  • the human cell is from a cultured cell line, for example such as can be obtained commercially. Exemplary cell lines include K562 cells, a human myelogenous leukemia line.
  • the cell is within a living organism. In certain other embodiments, the cell is outside of a living organism.
  • the method comprises contacting the DNA sequence with (i) a guide RNA or a set of guide RNA molecules described herein, and (ii) a Cas protein.
  • the method further comprises introducing or delivering the guide RNA into the cell.
  • the guide RNA is introduced into a cell by transfection. Techniques for RNA transfection are known in the art and include electroporation and lipofection.
  • the guide RNA is introduced into a cell (and, more particularly, a cell nucleus) by nucleofection. Techniques for nucleofection are known in the art and may utilize nucleofection devices such as the Lonza Nucleofector 2b or the Lonza 4D-Nucleofector and associated reagents.
  • the method further comprises introducing or delivering the Cas protein into the cell.
  • the Cas protein is introduced as a purified or non-purified protein.
  • the Cas protein is introduced via an mRNA encoding the Cas protein.
  • the mRNA encoding the Cas protein is introduced into the cell by transfection.
  • the mRNA encoding the Cas protein is introduced into a cell (and, more particularly, a cell nucleus) by nucleofection.
  • the method employs ribonucleoprotein (RNP)-based delivery such that the Cas protein is introduced into the cell in a complex with the guide RNA.
  • RNP ribonucleoprotein
  • a Cas9 protein may be complexed with a guide RNA in a Cas9:gRNA complex, which allows for co-delivery of the gRNA and Cas protein.
  • the Cas:gRNA complex may be nucleofected into cells.
  • the method employs an all-RNA delivery platform.
  • the guide RNA and the mRNA encoding the Cas protein are introduced into the cell simultaneously or substantially simultaneously (e.g., by co-transfection or co-nucleofection).
  • co-delivery of Cas mRNA and modified gRNA results in higher editing frequencies as compared to co-delivery of Cas mRNA and unmodified gRNA.
  • gRNA having 2′-O-methyl-3′-phosphorothioate (“MS”), 2′-O-methyl-3′-PACE (“MP”), or 2′-O-methyl-3′-thioPACE (“MSP”) incorporated at three terminal nucleotides at both the 5′ and 3′ ends provide higher editing frequencies as compared to unmodified gRNA.
  • the guide RNA and the mRNA encoding the Cas protein are introduced into the cell sequentially; that is, the guide RNA and the mRNA encoding the Cas protein are introduced into the cell at different times.
  • the time period between the introduction of each agent may range from a few minutes (or less) to several hours or days.
  • gRNA is delivered first, followed by delivery of Cas mRNA 4, 8, 12 or 24 hours later.
  • Cas mRNA is delivered first, followed by delivery of gRNA 4, 8, 12 or 24 hours later.
  • delivery of modified gRNA first, followed by delivery of Cas mRNA results in higher editing frequencies as compared to delivery of unmodified gRNA followed by delivery of Cas mRNA.
  • the gRNA is introduced into the cell together with a DNA plasmid encoding the Cas protein.
  • the gRNA and the DNA plasmid encoding the Cas protein are introduced into the cell by nucleofection.
  • an RNP-based delivery platform or an all-RNA delivery platform provides lower cytotoxicity in primary cells than a DNA plasmid-based delivery system.
  • the method provides significantly enhanced genome editing efficiencies in human cells, including human primary T cells and CD34+ HSPCs.
  • modified gRNA increases the frequency of insertions or deletions (indels), which may be indicative of mutagenic NHEJ and gene disruption, relative to unmodified gRNA.
  • modified gRNA having 2′-O-methyl-3′-phosphorothioate (“MS”), 2′-O-methyl-3′-PACE (“MP”), or 2′-O-methyl-3′-thioPACE (“MSP”) incorporated at three terminal nucleotides at both the 5′ and 3′ ends increases the frequency of indels relative to unmodified gRNA.
  • co-delivery of modified gRNA and Cas mRNA to human primary T cells increases the frequency of indels as compared to co-delivery of unmodified gRNA and Cas mRNA.
  • modified gRNA having 2′-O-methyl-3′-phosphorothioate (“MS”), 2′-O-methyl-3′-PACE (“MP”), or 2′-O-methyl-3′-thioPACE (“MSP”) incorporated at three terminal nucleotides at both the 5′ and 3′ ends increases the frequency of indels in human primary T cells relative to unmodified gRNA.
  • modified gRNA improves gRNA stability relative to unmodified gRNA.
  • gRNA having 2′-O-methyl (“M”) incorporated at three terminal nucleotides at both the 5′ and 3′ ends modestly improves stability against nucleases and also improves base pairing thermostability over unmodified gRNA.
  • gRNA having 2′-O-methyl-3′-phosphorothioate (“MS”), 2′-O-methyl-3′-PACE (“MP”), or 2′-O-methyl-3′-thioPACE (“MSP”) incorporated at three terminal nucleotides at both the 5′ and 3′ ends dramatically improves stability against nucleases relative to unmodified gRNA.
  • gRNA end modifications enhance intracellular stability against exonucleases, thus enabling increased efficacy of genome editing when Cas mRNA and gRNA are co-delivered or sequentially delivered into cells or cell lysates.
  • a stability-enhancing modification at an end may also serve as a specificity-enhancing modification if a guide sequence comprises the same end.
  • end modifications comprising 2′-O-methyl-3′-phosphonoacetate (MP), 2′-O-methyl-3′-thiophosphonoacetate (MSP), 2′-O-methyl-3′-phosphorothioate (MS), 2′-deoxy-3′-phosphonoacetate (DP), 2′-deoxy-3′-thiophosphonoacetate (DSP), 2′-fluoro-3′-phosphonoacetate (FP), 2′-fluoro-3′-thiophosphonoacetate (FSP), 2′-fluoro-3′-phosphorothioate (FS), 2′-O-(2-methoxyethyl)-3′-phosphonoacetate, 2′-O-(2-methoxyethyl)-thiophosphonoacetate, 2′-O-(2-methoxyethyl)-3′-phosphorothioate, or a combination thereof increases stability and specificity of a method of the present invention.
  • modified gRNA stimulates gene targeting, which, in turn, allows for gene editing by, for example, homologous recombination or NHEJ.
  • gRNA having 2′-O-methyl-3′-phosphorothioate (“MS”), 2′-O-methyl-3′-PACE (“MP”), or 2′-O-methyl-3′-thioPACE (“MSP”) incorporated at three terminal nucleotides at both the 5′ and 3′ ends stimulates higher levels of homologous recombination than unmodified gRNA.
  • modified gRNA retains high specificity.
  • the ratio of on-target to off-target indel frequencies is improved with modified gRNA as compared to unmodified gRNA.
  • modified gRNA delivered in an RNP complex with a Cas protein provides significantly better on-target versus off-target ratios compared to a DNA plasmid-based delivery system.
  • the guide RNA described herein is used for regulating transcription or expression of a gene of interest.
  • a fusion protein comprising a Cas protein (e.g., a nuclease-deficient Cas9) and a transcription activator polypeptide is used to increase transcription of a gene.
  • a fusion protein comprising a Cas protein (e.g., a nuclease-deficient Cas9) and a repressor polypeptide is used to knock-down gene expression by interfering with transcription of the gene.
  • the present invention provides a method for regulating the expression of a gene of interest in vivo or in vitro.
  • the method comprises introducing into a cell or another system (i) a synthetic guide RNA described herein, and (ii) a fusion protein.
  • the fusion protein comprises a Cas protein and an effector domain, such as a transcriptional activation domain, a transcriptional repressor domain, or an epigenetic modification domain.
  • the fusion protein comprises a mutated Cas protein, such as a Cas9 protein that is a null nuclease.
  • the Cas protein comprises one or more mutations, such as D10A, H840A and/or N863A.
  • the fusion protein is introduced into the cell or system as a purified or non-purified protein. In certain embodiments, the fusion protein is introduced into the cell or system via an mRNA encoding the fusion protein. In certain embodiments, the fusion protein is introduced into the cell or system via a linear or circular DNA encoding the fusion protein.
  • the guide RNA aids in directing the fusion protein to a specific target polynucleotide comprising a chromosomal sequence, an episomal sequence, a plasmid, a mitochondrial DNA sequence, or a functional intergenic sequence, such as an enhancer or the DNA sequence for a non-coding RNA.
  • the effector domain regulates expression of a sequence in the target polynucleotide.
  • a guide RNA for modulating gene expression can be designed to target any desired endogenous gene or sequence encoding a functional RNA.
  • a genomic target sequence can be selected in proximity of the transcription start site of the endogenous gene, or alternatively, in proximity of the translation initiation site of the endogenous gene.
  • the library targets at least 10,000 different polynucleotides or at least 10,000 different sequences within one or more polynucleotides. In certain embodiments, the library targets at least 100,000 different polynucleotides or at least 100,000 different sequences within one or more polynucleotides. In certain embodiments, the library targets at least 1,000,000 different polynucleotides or at least 1,000,000 different sequences within one or more polynucleotides.
  • the present invention provides a kit for selecting a synthetic guide RNA comprising at least two synthetic guide RNAs which are identical except for different modifications or modifications at different positions in the guide sequence.
  • Each guide RNA comprises (a) a crRNA segment comprising (i) a guide sequence capable of hybridizing to a target polynucleotide, (ii) a stem sequence; and (b) a tracrRNA segment comprising a nucleotide sequence that is partially or completely complementary to the stem sequence, wherein the guide sequence comprises nucleotides 1 through 20-N (N is an integer between ⁇ 10 and 10, optionally between ⁇ 10 and 6), counted from the 5′ end of the guide sequence, and at least one specificity-enhancing modification at a nucleotide in the guide sequence; wherein the at least two synthetic guide RNAs differ from each other by having at least one different specificity-enhancing modification or by having the specificity-enhancing modification at least one different position in the guide sequence.
  • the kit also comprises a Cas protein or a polynucleotide coding for a Cas protein.
  • each synthetic guide RNA in the kit comprises a specificity-enhancing modification at a different nucleotide.
  • the kit comprises a series of synthetic guide RNAs, each one having a modification at a different nucleotide position in the guide sequence.
  • the kit has the same number of different guide RNAs as the number of nucleotides in the guide sequence.
  • the Cas protein is Cas9.
  • the specificity-enhancing modification comprises 2′-O-methyl-3′-phosphonoacetate (MP), 2′-O-methyl-3′-thiophosphonoacetate (MSP), 2′-deoxy-3′-phosphonoacetate (DP), or 2′-deoxy-3′-thiophosphonoacetate (DSP), or a combination thereof.
  • the specificity-enhancing modifications comprise a 2′-modification that confers a C3′-endo sugar pucker and a phosphonoacetate or thiophosphonoacetate linkage modification.
  • the 2′-modification is selected from 2′-F and 2′-O-(2-methoxyethyl).
  • the guide RNAs are synthetic single guide RNAs.
  • kits examples include, but are not limited to, one or more different polymerases, one or more host cells, one or more reagents for introducing foreign nucleic acid into host cells, one or more reagents (e.g., probes or PCR primers) for detecting expression of the guide RNA and/or the Cas mRNA or protein or for verifying the status of the target nucleic acid, and buffers, transfection reagents or culture media for the reactions (in 1 ⁇ or more concentrated forms).
  • the kit includes one or more of the following components: biochemical and physical supports; terminating, modifying and/or digesting reagents; osmolytes; and apparati for reaction, transfection and/or detection.
  • the reaction components used can be provided in a variety of forms.
  • the components e.g., enzymes, RNAs, probes and/or primers
  • the components can be suspended in an aqueous solution or bound to a bead or as a freeze-dried or lyophilized powder or pellet.
  • the components when reconstituted, form a complete mixture of components for use in an assay.
  • the kits of the invention can be provided at any suitable temperature.
  • the container(s) in which the components are supplied can be any conventional container that is capable of holding the supplied form, for instance, microfuge tubes, microtiter plates, ampoules, bottles, or integral testing devices, such as fluidic devices, cartridges, lateral flow, or other similar devices.
  • kits can also include packaging materials for holding the container or combination of containers.
  • packaging materials for such kits and systems include solid matrices (e.g., glass, plastic, paper, foil, micro-particles and the like) that hold the reaction components or detection probes in any of a variety of configurations (e.g., in a vial, microtiter plate well, microarray, and the like).
  • the kits may further include instructions recorded in a tangible form for use of the components.
  • a 43-nucleotide crRNA with a 20-nucleotide guide sequence was made.
  • a duplex was formed by mixing the crRNA with a complementary 40-nucleotide DNA oligonucleotide that comprised a 10 nucleotides overhanging on each end of the DNA oligonucleotide.
  • the melting temperature (“Tm”) of the duplex was measured.
  • Seven additional 43-nt crRNAs were made, with several sequential modifications in the sampling and locking region and an intervening or “spacer” nucleotide at position 10 in the 20-nt guide sequence.
  • FIG. 6A shows the type and placement of the modifications.
  • FIG. 6B shows the melting curve for unmodified gRNA
  • FIGS. 6C through 6G show melting curves for various types of modifications at nucleotides 6 through 9 in the 20-nt guide sequence portions.
  • M 2′-O-methyl
  • MS 2′-O-methyl phosphorothioate
  • FIG. 7 is a graph showing change in melting temperature of a 20-base pair gRNA/DNA duplex as the gRNA was truncated to 17, 18 or 19 nucleotides or extended to 21 or 22 nucleotides in length. The measurements were performed in physiological salt concentrations. The measurements indicated a change in melting temperature of about 2° C. per base pair for the extension or truncation of the 20-nucleotide guide sequence.
  • the linker or linker-like modifications comprised a ULNA (unlocked nucleic acid), an abasic spacer, an alkylene spacer comprising —PO 4 Y—(CR 3 2 ) m -PO 4 Y—, or an ethylene glycol spacer comprising (—PO 4 Y—(CR 3 2 CR 3 2 O) u —PO 3 Y—), where m is 2, 3 or 4, n is 1, 2 or 3, each R 3 is independently selected from the group consisting of H, an alkyl and substituted alkyl, and each Y is H or a negative charge.
  • ULNA locked nucleic acid
  • an abasic spacer an alkylene spacer comprising —PO 4 Y—(CR 3 2 ) m -PO 4 Y—
  • an ethylene glycol spacer comprising (—PO 4 Y—(CR 3 2 CR 3 2 O) u —PO 3 Y—)
  • the alkylene spacer is a “C3 spacer” in which m is 3.
  • the ethylene glycol spacer is a “Tri-PEG spacer” in which n is 3.
  • PAM-addressable DNA constructs comprising target polynucleotide sequences (ON) or off-target polynucleotide sequences (OFF) set forth in Tables 2 and 7 were prepared by preparative PCR amplification of plasmid-borne human sequences of various target genes.
  • the human clathrin light chain CLTA gene As exemplary genes to demonstrate the ability of the present compositions and methods to selectively edit genes, the human clathrin light chain CLTA gene, the human hemoglobin beta (HBB) gene, the human interleukin 2 receptor subunit gamma (IL2RG) gene, and the human cytotoxic T-lymphocyte-associated protein 4 (CLTA4) gene were used as target genes. These are representative of the general approach disclosed herein for evaluating and editing target genes.
  • Tables 3 to 6 set forth synthetic guide RNAs.
  • the first 20 nucleotides at the 5′ end are complementary to the target sequence in target DNA—these complementary nucleotides make up the guide sequence.
  • an overhang or extension was present at the 5′ end of the guide sequence, which is not complementary to the target sequence.
  • the 5′ end of the guide sequence was truncated such that nucleotides 1, or 1 and 2, or 1 and 2 and 3, are not present.
  • On-target constructs (“ON”) comprise the 20-nt target sequence.
  • Off-target constructs (“OFF”) comprise most of the same 20 nucleotides as the target DNA, with 1, 2 or 3 nucleotide differences. Accordingly, the gRNA was mostly, but not completely, complementary to the sequence of the OFF target constructs. The OFF target constructs are based on gene sequences known to occur in the human genome.
  • gRNAs were synthesized on an ABI 394 Synthesizer (Life Technologies, Carlsbad, Calif., USA) using 2′-O-thionocarbamate-protected nucleoside phosphoramidites according to procedures described in Dellinger et al. (2011) J. Am. Chem. Soc., 133, 11540-56. 2′-O-methyl phosphoramidites were incorporated into RNA oligomers under the same conditions as the 2′-O-thionocarbamate protected phosphoramidites.
  • RNAs 2′-O-methyl-3′-O-(di-iso-propylamino)phosphinoacetic acid-1,1-dimethylcyano-ethyl ester-5′-O-dimethoxytrityl nucleosides used for synthesis of thiophosphonoacetate (thioPACE)-modified RNAs were synthesized essentially according to published methods. See Dellinger et al. (2003) J. Am. Chem. Soc. 125, 940-50; and Threlfall et al. (2012) Org. Biomol. Chem. 10, 746-54.
  • thioPACE thiophosphonoacetate
  • oligonucleotides were purified using reversed-phase high-performance liquid chromatography (HPLC) and analyzed by liquid chromatography-mass spectrometry (LC-MS) using an Agilent 1290 Infinity series LC system coupled to an Agilent 6520 Q-TOF (time-of-flight) mass spectrometer (Agilent Technologies, Santa Clara, Calif., USA).
  • the yields for the synthesis and purification of the sgRNAs were estimated using deconvolution of mass spectra obtained from LC-MS-derived total ion chromatograms.
  • the chemical synthesis of the 100-mer sgRNAs typically yielded 25-35% full-length product from a nominal 1 micromole scale synthesis.
  • Reversed-phase HPLC purification using ion pairing buffer conditions typically gave 20% yield from the crude product with an estimated purity of the final sgRNA in the range of 90% to 95%.
  • the DNA target constructs comprised the target sequences (also known as on-target sequences or identified as “ON”) and off-target sequences (“OFF”) set forth in Table 2, with differences in the off-target sequences from the target in bold italics, and the PAM sequences (when shown) are underlined:
  • HBB OFF1 differs from HBB ON in the first 3 nucleotides of the potential target sequence. Therefore, a gRNA truncated at the 5′ end of its 20-nt guide sequence by 3 nucleotides to provide a 17-nt guide sequence cannot distinguish between HBB—ON and HBB—OFF1 target sequences.
  • Table 8 The full sequence of the DNA target constructs used in Examples 3-7 is set forth in Table 8 below.
  • RNAs having truncation of the guide sequence of a gRNA from 20 nucleotides to 18 or 17 nucleotides, with an evaluation of the ratio of cleavage of target gene sites to known off-target sites. It was seen that, in contrast to the teachings and conclusion by Y searching et al. (2014), truncation had an effect on cleavage of specific off-target sites but did not have an effect on cleavage at other off-target sites. In spite of the teachings of Ysweeping et al. (2014), the present inventors have sought and identified novel compounds and methods for CRISPR-Cas cleaving, nicking or binding a target polynucleotide with enhanced specificity and without truncation of the guide sequence.
  • Crude products were loaded into a DNA 1000 or DNA 7500 LabChip for analysis on an Agilent Bioanalyzer 2200 or were loaded onto a Genomic DNA ScreenTape or a D5000 ScreenTape for analysis on an Agilent TapeStation 2200 or 4200.
  • the workup steps serve to release Cas9 from binding of target DNA, which was assayed for cleavage.
  • Cleavage yields were calculated by the formula: a/(a+b) ⁇ 100 where a is the sum of the band intensities of the two cleavage products and b is the remaining uncleaved DNA if present.
  • a cleavage percentage of 100% means that all of the target DNA construct was cleaved, within the limits of detection.
  • RNA strands were synthesized and HPLC purified. All oligonucleotides were quality control approved on the basis of full-length strand purity by HPLC analysis and chemical composition by mass spectrometry analysis.
  • Table 3 sets forth the sequences of the various CLTA1 sgRNAs. Table 3 shows the sequences of sgRNAs as Entries 1 through 31, and the table discloses certain embodiments of the present gRNAs containing one or more specificity-enhancing modifications. Entry 1 was unmodified and serves as a comparative example. Entry 2 contains MS modifications in the guide sequence at nucleotides 1, 2 and 3.
  • Entry 3 contains MSP modifications in the guide sequence at nucleotides 1, 2 and 3.
  • Entry 4 contains an MSP modification at nucleotide 1 of the guide sequence.
  • Entries 5 and 6 are comparable examples having a gRNA truncated at the 5′ end of its 20-nt guide sequence to an 18-nucleotide guide sequence or a 17-nucleotide guide sequence, respectively.
  • Entries 7 and 8 are comparable examples having unmodified gRNA with one- or two-nucleotide overhangs, respectively, at the 5′ end of the 20-nt guide sequence.
  • Entries 9, 10, 11, 12, and 13 contain MP modifications in the guide sequence at nucleotide 1, nucleotides 1-2, nucleotides 1-3, nucleotides 1-4, and nucleotides 1-5, respectively.
  • Entries 14 and 15 contain MP modifications in the tracrRNA region of the gRNA at nucleotides 2-5 counted from the 3′ end of the sgRNA or nucleotides 2-6 counted from the 3′ end of the sgRNA, respectively, noting that nucleotides are generally counted from 5′ ends of polynucleotides. Therefore, the counting described for entries 14 and 15 is an exception to the general rule.
  • Entries 16 and 17 contain MP modifications in the 20-nt guide sequence at nucleotides 1-2, with an MP-modified C or G nucleotide overhang, respectively.
  • Entries 18 and 19 contain MP modifications in the 20-nt guide sequence at nucleotides 1-3, with an MP-modified UC or AG dinucleotide overhang, respectively.
  • Entries 20 and 21 contain MP modifications in the 20-nt guide sequence at nucleotides 1-4, with an MP-modified CUC or GAG trinucleotide overhang, respectively, plus MP modifications in the tracrRNA region of the gRNA at the 3′-end of the sgRNA.
  • Entries 22-25 contain MP modification in the guide sequence at nucleotide 20, 19, 18 or 17, respectively.
  • Entry 26 contains MP modifications in the guide sequence at nucleotides 18 and 17.
  • Entries 27-29 contain an M modification in the guide sequence at nucleotide 19, 18 or 17, respectively.
  • Entry 30 contains M modifications in the guide sequence at nucleotides 18 and 17.
  • Entry 31 contains M modifications in the guide sequence at nucleotides 1-20.
  • Entry 32 contains M modifications in the 20-nt guide sequence at nucleotides 1-7, 9-11, 13-14 and 20, plus M modifications at several select positions across the remainder of the sgRNA sequence, specifically at nucleotides 30-31, 33, 35-36, 39, 42, 45, 47, 50, 60, 65-66, 70, 71, 76-77, 80-82, 90, 93, 95-96, 100-101, 104, and 106-112.
  • FIG. 8A shows the impact of chemical modifications in the gRNAs from Table 3 (SEQ ID NO: 12-42 and 124) with regard to Cas9-mediated target polynucleotide cleavage versus off-target polynucleotide cleavage. More particularly, the cleavage percentages of a CLTA1 target polynucleotide sequence (the on-target sequence, or “ON”) and comparable off-target polynucleotide sequences (OFF1 and OFF3) are shown numerically and in bar graph form.
  • FIG. 8B is derived from the results in FIG.
  • RNA strands were synthesized and HPLC purified. All oligonucleotides were quality control approved on the basis of full-length strand purity by HPLC analysis and chemical composition by mass spectrometry analysis.
  • the cleavage percentages of a CLTA4 target polynucleotide sequence (ON) and comparable off-target polynucleotide sequences (OFF1, OFF2 and OFF3) are shown numerically and in bar graph form in FIG. 9A .
  • FIG. 9B is derived from the results in FIG.
  • Shading indicates which MP positions in the guide sequence provided at least two-fold improvements in specificity.
  • the results of the MP walk in entries 1-18 indicate that placement of the walked MP modification has an effect on specificity, and a trend is apparent for each set of specificity scores per off-target site assayed which shows that an MP modification near the 5′ end of the guide sequence enhances specificity more so than an MP at other positions in the guide sequence, as seen in entries 1 and 2 relative to entries 3-18.
  • This trend is consistent with a specificity enhancement trend observed for MP modifications in gRNAs targeted to the CLTA1 target sequence, in which MP modifications added to the 5′ end of the CLTA sgRNA enhanced specificity, as indicated by the shaded scores in entries 12-13 and 20-21 in FIG. 8B .
  • a general strategy for improving specificity is to incorporate 1, 2, 3, 4 or 5 MP modifications at consecutive phosphodiester internucleotide linkages at the 5′ end of a guide sequence in a gRNA.
  • the experimental sgRNAs contained 2′-O-methyl-3′-PACE (“MP”) modification at one or more positions in the guide sequence, in addition to having an MP modification at nucleotide 1 and also at the penultimate nucleotide in the tracrRNA region at the 3′ end of the sgRNA which includes the last (i.e., most 3′) internucleotide linkage.
  • MP 2′-O-methyl-3′-PACE
  • the cleavage percentages of an IL2RG target polynucleotide sequence (ON) and a comparable off-target polynucleotide sequence (OFF3) are shown numerically in FIG. 10 .
  • the figure also shows a ratio calculated for cleaved target polynucleotide versus cleaved off-target polynucleotide for each synthetic sgRNA (SEQ ID NO: 71-86 and 173-186) assayed.
  • a Specificity Score is calculated by multiplying a ratio by its respective on-target cleavage percentage.
  • Specificity scores ⁇ 2.0 are shaded to indicate an improvement in specificity relative to unshaded scores, therefore the shading indicates which MP positions in the guide sequence provided at least two-fold improvements in specificity.
  • FIG. 10 the positions in the guide sequence which yielded the greatest improvements in specificity are indicated by darker shading.
  • the results from MP modification of position 7, 14 or 16 are shown in entries 6, 13 and 15, respectively.
  • Guide RNAs possessing such compositions can enhance specificity performance relative to other commonly used compositions such as gRNAs lacking MP modifications, particularly for various CRISPR-Cas applications of gRNAs targeting the clinically important IL2RG locus.
  • the example represented by FIG. 10 also instantiates the present novel method of “walking” an MP modification across the guide sequence portion of a gRNA to identify which position or positions yield specificity enhancement due to the location of the walked MP modification.
  • the magnitude of the specificity enhancement is assessed by the on-target versus off-target cleavage ratio for each position tested, and a practitioner can consider such values alongside the percentage of on-target cleavage measured for each design when deciding which MP-modified position or positions is likely to benefit the overall performance of the gRNA.
  • the incremental walking of a single MP across the guide sequence may also identify positions in the sequence for potential synergistic improvements in specificity resulting from one or more combinations of MP modifications at the positions tested by the walk.
  • a modification “walk” was done with an MP modification installed at incremental positions across a guide sequence targeted to the human HBB gene as shown in FIG. 11A to see if various sites in the 20-nt guide sequence may give substantial cleavage of the on-target site and decreased cleavage of the off-target site.
  • sgRNAs were made for targeting the HBB gene as listed in Table 6, in which experimental sgRNAs contained a 2′-O-methyl-3′-PACE (“MP”) modification at one or more internal nucleotide positions in the guide sequence, in addition to having an MP modification at nucleotide 1 and also at the penultimate nucleotide in the tracrRNA region at the 3′ end of the sgRNA which includes the last (i.e., most 3) internucleotide linkage.
  • MP 2′-O-methyl-3′-PACE
  • the modifications in the terminal internucleotide linkages were designed to protect the sgRNAs against degradation by exonucleases. Individual RNA strands were synthesized and HPLC purified.
  • oligonucleotides were quality control approved on the basis of full-length strand purity by HPLC analysis and chemical composition by mass spectrometry analysis.
  • the cleavage percentages of an HBB target polynucleotide sequence (ON) and a comparable off-target polynucleotide sequence (OFF1) are shown numerically in FIG. 11A .
  • the figure also shows a ratio calculated for cleaved target polynucleotide versus cleaved off-target polynucleotide for each synthetic sgRNA (SEQ ID NO: 87-103) assayed.
  • a Specificity Score is calculated by multiplying a ratio by its respective on-target cleavage percentage.
  • Specificity scores ⁇ 2.0 are shaded to indicate an improvement in specificity relative to unshaded scores, therefore the shading indicates which MP positions in the guide sequence provided at least two-fold improvements in specificity.
  • FIG. 11A the positions in the guide sequence that yielded the greatest improvements in specificity are indicated by darker shading, resulting from MP modification of positions 5, 9 or 11 as shown in entries 2, 6 and 8, respectively.
  • Guide RNAs possessing such compositions can enhance specificity relative to other commonly used compositions, such as gRNAs lacking MP modifications, particularly for various CRISPR-Cas applications of gRNAs targeting the clinically important HBB locus.
  • 11A also instantiates our method of “walking” an MP modification across the guide sequence portion of a gRNA to identify which position or positions yield specificity enhancement due to the location of the walked MP modification.
  • the magnitude of the specificity enhancement is assessed by the on-target versus off-target cleavage ratio for each position tested, and a practitioner can consider such values alongside the percentage of on-target cleavage measured for each design when deciding which MP-modified position or positions is likely to benefit the overall performance of the gRNA.
  • the incremental walking of a single MP across the guide sequence may also identify positions in the sequence for potential synergistic improvements in specificity resulting from one or more combinations of MP modifications at the positions tested by the walk.
  • FIG. 11B shows the impact of various types of modifications in sgRNAs targeting the human HBB gene in K562 cells by co-transfecting each synthetic sgRNA (SEQ ID NO: 187-190) with Cas9 mRNA and measuring cleavage of the target locus and three off-target loci including the same off-target sequence OFF1 as evaluated in FIG. 11A .
  • a ratio is calculated for cleaved target (ON) versus cleaved off-target polynucleotide (OFF1) for each synthetic sgRNA assayed.
  • a Specificity Score is calculated by multiplying a ratio by its respective on-target cleavage percentage. The results show that the PACE modifications in the guide sequence yielded a substantial improvement in specificity as evaluated by the Specificity Scores for the various types of modifications tested, especially with respect to the primary off-target activity (at OFF1).
  • a ratio was calculated for cleaved target polynucleotide (ON) versus cleaved off-target polynucleotide (OFF1) for each synthetic sgRNA assayed.
  • a Specificity Score was calculated by multiplying a ratio by its respective on-target cleavage percentage. Entries 18-64 are ranked according to Specificity Score from highest to lowest. Entries with the highest scores are shaded.
  • FIG. 12B shows the results of editing a genomic HBB target which has the same 20-base pair sequence as was tested in polynucleotide constructs in vitro for FIG. 12A .
  • the genomic HBB target site is endogenous in the human K562 cells and iPS cells tested.
  • the results are grouped according to the number of MP modifications incorporated in the 20-nt guide sequence. Entries 1-17 in FIG. 12A and entries 1-12 and 22-33 in FIG.
  • each sgRNA tested were SEQ ID NO: 88, 90, 92-94, 96, 97, 99, 100, 103, 171 and 172.
  • the sgRNA tested were 128, 129, 133, 141, 160, 165, and 168.
  • Results for a pair of internal MP modifications are grouped separately in entries 18-64 in FIG. 12A and in entries 13-19 and 34-40 in FIG. 12B , and the relative performance of these designs in vitro and in vivo as ranked by Specificity Score is remarkably consistent across the various assays and cell types.
  • a slightly different way of evaluating specificity enhancement is simply to consider the on-target versus off-target ratio, which is ON:OFF1 for the HBB examples in FIGS. 12A and 12B .
  • a re-ranking of the groupings in FIG. 12B according to measured ratio, sorted from highest to lowest ratio per grouping, is shown in FIG. 12C .
  • the various groupings in the figure are separated by heavy black lines.
  • FIG. 14 shows the impact of various types of modifications in IL2RG sgRNAs and VEGFA sgRNAs in K562 cells.
  • Each synthetic sgRNA was co-transfected with Cas9 mRNA, and cleavage of the target locus and of an off-target locus OFF2 were measured.
  • a ratio is calculated for cleaved target (ON) versus cleaved off-target polynucleotide (OFF2) for each synthetic sgRNA assayed.
  • a Specificity Score is calculated by multiplying a ratio by its respective on-target cleavage percentage.
  • FIG. 14 show that modifications in the guide sequence of synthetic gRNAs targeting IL2RG and VEGFA yielded substantial improvements in specificity as evaluated by the Specificity Scores for the various types of modifications tested, especially with respect to the off-target activity (at OFF2).
  • entry 1 shows a significant specificity enhancement resulted from MP modification at both ends of the IL2RG sgRNA, in which the 5′ and 3′ terminal internucleotide linkages are modified.
  • entries 3 & 4 show impressive specificity enhancements for IL2RG sgRNAs having an MP modification at an internal position in the IL2RG guide sequence, namely at position 5 or 11, respectively.
  • MP modification of position 5 gave the largest enhancement of specificity among entries 1 thru 4 in FIG. 14 .
  • entries 6 thru 10 show significant specificity enhancements for VEGFA sgRNA having an MP modification at an internal position in the VEGFA guide sequence, namely at position 5, 7, 9, 10, or 11, respectively.
  • entries 5 thru 10 show significant specificity enhancements for VEGFA sgRNA having an MP modification at an internal position in the VEGFA guide sequence, namely at position 5, 7, 9, 10, or 11, respectively.
  • FIG. 13 shows a composite map of MP-modified positions in various guide sequences which yielded specificity enhancements.
  • FIG. 13 shows a comparison of in vitro cleavage results from FIGS. 9A, 9B, 10 and 11A .
  • FIG. 13 shows several important trends. First, as shown in Entry 12 in contrast to all other entries in FIG. 13 , gRNAs having an MP modification at position 15 in their 20-nt guide sequences suffered a substantial loss of Cas9-mediated cleavage activity.
  • Example 6 A further example of the utility of the general approach was discussed above in Example 6 regarding MSP modifications at the 5′ end of a gRNA targeting the HBB gene in transfected human K562 cells as shown in FIG. 11B .
  • a third example regarding specificity effects of MP modifications is apparent in entry 3 of FIG. 13 , where specificity scores resulting from MP modification of position 6 in differently targeted 20-nt guide sequences constitute the lowest scores per MP walk, disregarding the anomalous effects for position 15 mentioned above.
  • a nadir of specificity enhancement due to MP modification of position 6 is especially noticeable in contrast to specificity scores resulting from MP modification of an adjacent position in the same guide sequence, namely position 4, 5 or 7 (shown as shaded scores throughout entries 1, 2 or 4, respectively).
  • Examples 3 through 7 demonstrate that gRNAs containing modifications at specific positions in guide sequences are tolerated by active Cas protein and gRNA:Cas protein complexes, as modifications at many sequence positions in gRNAs did not prevent target-specific cleavage of on-target polynucleotides.
  • Examples 3 through 7 also demonstrate that the present gRNAs, complexes and methods can achieve on-target versus off-target ratios of at least 1.2, alternatively at least 1.5, alternatively at least 2, alternatively at least 2.5, alternatively at least 3, alternatively at least 3.5, alternatively at least 4, alternatively at least 4.5, alternatively at least 5, and/or the ratio is up to 10, 12, 15 or 20.
  • the 2′-O-methyl-3′-PACE (“MP”) modification had a positive effect on ratios by decreasing off-target cleavage levels while retaining high levels of on-target cleavage as desired for specificity enhancements.
  • a synthetic guide RNA comprising:
  • a crRNA segment comprising (i) a guide sequence capable of hybridizing to a target polynucleotide, (ii) a stem sequence;
  • A6 The synthetic guide RNA of any of the preceding embodiments, wherein the guide sequence comprises a locking region, a sampling region, and a seed region, and the at least one specificity-enhancing modification is present in the sampling region and/or in the seed region.
  • A9 The synthetic guide RNA of any of the preceding embodiments, wherein the at least one specificity-enhancing modification comprises a chemically modified nucleobase.
  • the synthetic guide RNA of any of the preceding embodiments comprising a 2′ modification that confers a C3′-endo sugar pucker.
  • A15 The synthetic guide RNA of any of the preceding embodiments, wherein the at least one specificity-enhancing modification is an unstructured nucleic acid (“UNA”), an unlocked nucleic acid (‘ULNA’), an abasic nucleotide, or an alkylene spacer comprising —PO 4 Y—(CR 3 2 )m-PO 4 Y—, or an ethylene glycol spacer comprising (—PO4Y-(CR 3 2 CR 3 2 O)n-PO 3 Y—), where m is 2, 3 or 4, n is 1, 2 or 3, each R 3 is independently selected from the group consisting of H, an alkyl and a substituted alkyl, and each Y is H or a negative charge.
  • UNA unstructured nucleic acid
  • ULNA unlocked nucleic acid
  • abasic nucleotide or an alkylene spacer comprising —PO 4 Y—(CR 3 2 )m-PO 4 Y—, or an ethylene glycol spacer
  • A16 The synthetic guide RNA of any of embodiments A1 to A9, A11 to A13, or A1 S, wherein the at least one specificity-enhancing modification does not comprise a nucleobase modification.
  • A17 The synthetic guide RNA of any of embodiments A1 to A15, wherein the at least one specificity-enhancing modification is a nucleobase selected from the group consisting of 2-thioU, 2-thioC, 4-thioU, 6-thioG, 2-aminopurine, hypoxanthine, 7-deazaguanine, 7-deaza-8-azaguanine, 7-deazaadenine, 7-deaza-8-azaadenine, 5-methylC, 5-methylU, 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5,6-dehydrouracil, 5-ethynylcytosine, 5-aminoallylU, 5-aminoallylC, an abasic nucleotide, a UNA base, isoC, isoG, 5-methyl-pyrimidine, x(A,G,C,T,U), y(A,G,C,T,U), and combinations thereof.
  • the at least one specificity-enhancing modification is a nucleobase selected from
  • A18 The synthetic guide RNA of any of the preceding embodiments, wherein the at least one specificity-enhancing modification is a nucleotide or nucleotide analog selected from the group consisting of 5-nitroindole, nebularine, inosine, diaminopurine, an abasic linkage, and an abasic fluorophore linkage such as 3-O-yl-2-(4-butylamidofluorescein)propyl-1-O-yl-phosphodiester.
  • the at least one specificity-enhancing modification is a nucleotide or nucleotide analog selected from the group consisting of 5-nitroindole, nebularine, inosine, diaminopurine, an abasic linkage, and an abasic fluorophore linkage such as 3-O-yl-2-(4-butylamidofluorescein)propyl-1-O-yl-phosphodiester.
  • A19 The synthetic guide RNA of any of the preceding embodiments, wherein the at least one specificity-enhancing modification comprises a modification that lowers a melting temperature (Tm) of a first DNA/RNA duplex formed by the synthetic guide RNA and the target polynucleotide, relative to the Tm of a duplex without the specificity-enhancing modification.
  • Tm melting temperature
  • the synthetic guide RNA of embodiment A21 wherein the guide sequence comprises or consists of nucleotides 1 through 20, counted from the 5′ end of the guide sequence, and at least one specificity-enhancing modification at nucleotide 1, alternatively at nucleotides 1 and 2, alternatively at nucleotides 1, 2, and 3, alternatively at nucleotides 1, 2, 3 and 4; alternatively at nucleotides 1, 2, 3, 4 and 5.
  • the synthetic guide RNA of embodiment A21 wherein the guide sequence consists of nucleotides 1 through 20-N, counted from the 5′ end of the guide sequence, where N is an integer between ⁇ 10 and 10 (optionally between ⁇ 10 and 6), and the at least one specificity-enhancing modification is within nucleotides 4-N to 20-N, alternatively within nucleotides 5-N to 20-N, alternatively within nucleotides 10-N to 20-N, alternatively within nucleotides 13-N to 20-N, alternatively within nucleotides 13-N through 14-N or 16-N through 19-N, alternatively within nucleotides 13-N through 14-N or 16-N through 18-N.
  • the synthetic guide RNA of embodiment A21 wherein the guide sequence consists of nucleotides 1 through 20-N, wherein N is a positive or negative integer between ⁇ 10 and 10 (optionally between ⁇ 10 and 6), counted from the 5′ end of the guide sequence, and the guide sequence comprises one specificity-enhancing modification located at nucleotide 11-N, 12-N, 13-N, 14-N, 16-N, 17-N, 18-N, 19-N or 20-N, alternatively located at nucleotide 13-N, 14-N, 16-N, 17-N, 18-N, 19-N or 20-N, alternatively located at nucleotide 13-N, 14-N, 16-N, 17-N, 18-N, 19-N or 20-N, alternatively located at nucleotide 13-N, 14-N, 16-N, 17-N, or 18-N.
  • a method of preparing a synthetic guide RNA comprising:
  • the off-target polynucleotide comprises a respective nucleotide sequence
  • off-target polynucleotide is identified by an algorithm to predict off-target sites such as those found at http://www.rgenome.net/Cas-OFFinder, https://cm.jefferson.edu/Off-Spotter, or http://crispr.mit.edu, or other technique for identifying and quantifying the activation of off-target sites in actual cases, such as disclosed in Tsai et al. (2015) Nat. Biotechnol. 33, 187-97; Ran et al. (2015) Nature 520, 186-91; and/or Frock et al. (2015) Nat. Biotechnol. 33, 179-86.
  • Tm of the first DNA/RNA duplex is higher than the Tm of the second DNA/RNA duplex, for example at least at least about 0.5° C. higher, alternatively at least about P° C. higher.
  • a crRNA segment comprising (i) a guide sequence capable of hybridizing to a target polynucleotide, (ii) a stem sequence;
  • a tracrRNA segment comprising a nucleotide sequence that is partially or completely complementary to the stem sequence
  • the guide sequence includes a specificity-enhancing modification
  • Tm melting temperature
  • gRNA:Cas protein complex comprising a Cas protein and the synthetic guide RNA
  • a method for cleaving, nicking or binding a target polynucleotide comprising:
  • a crRNA segment comprising (i) a guide sequence capable of hybridizing to a target polynucleotide, (ii) a stem sequence;
  • a tracrRNA segment comprising a nucleotide sequence that is partially or completely complementary to the stem sequence
  • gRNA:Cas protein complex comprising a Cas protein and the designed synthetic guide RNA
  • contacting the target polynucleotide with a gRNA:Cas protein complex at a temperature within 12° C. of the Tm, alternatively within 8° C. of the Tm, alternatively within 5° C. of the Tm, alternatively at approximately the Tm;
  • a method for cleaving, nicking or binding a target polynucleotide comprising:
  • Tm melting temperature
  • gRNA:Cas protein complex comprising a Cas protein and the designed synthetic guide RNA
  • contacting the target polynucleotide with a gRNA:Cas protein complex at a temperature within 12° C. of the Tm, alternatively within 8° C. of the Tm, alternatively within 5° C. of the Tm, alternatively at approximately the Tm;
  • EFG2 The method of any of the preceding embodiments E1, F1 or G1, wherein the at least one specificity-enhancing modification weakens hybridization between the guide sequence and the target polynucleotide.
  • EFG3 The synthetic guide RNA of any of embodiments E1, F1 or G1, wherein the at least one specificity-enhancing modification weakens hybridization between the guide sequence and an off-target polynucleotide.
  • EFG4 The synthetic guide RNA of any of embodiments E1, F1 or G1, wherein the at least one specificity-enhancing modification strengthens hybridization between the guide sequence and the target polynucleotide and weakens hybridization between the guide sequence and an off-target polynucleotide.
  • exogenous or endogenous template polynucleotide comprises at least one sequence having substantial sequence identity with a sequence on either side of the cleavage site.
  • a set or library of synthetic guide RNA molecules comprising two or more synthetic guide RNAs of any of the preceding embodiments.
  • kits comprising a synthetic guide RNA of any of the preceding embodiments, and one or more other components.
  • RNA molecules comprising two or more synthetic guide RNAs of any of the preceding embodiments.
  • the synthetic guide RNA, method, set or library, kit or array of any of the preceding embodiments comprising a single RNA strand that comprises both the crRNA segment and the tracrRNA segment.
  • L4A The synthetic guide RNA, method, set or library, kit or array of embodiment L3, wherein the loop L comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.
  • a method for cleaving, nicking or binding a target HBB polynucleotide comprising:
  • RNA comprising:
  • a crRNA segment comprising (i) a guide sequence capable of hybridizing to a target HBB polynucleotide, (ii) a stem sequence;
  • a tracrRNA segment comprising a nucleotide sequence that is partially or completely complementary to the stem sequence
  • the guide sequence consists of 20-N nucleotides, where N is an integer between ⁇ 10 and 10, and the guide sequence comprises at least one specificity-enhancing modification at a nucleotide selected from positions 4-N, 5-N, 7-N, 9-N, 10-N, and 11-N; and
  • gRNA:Cas protein complex comprising a Cas protein and the synthetic guide RNA
  • a synthetic guide RNA comprising:
  • a crRNA segment comprising (i) a guide sequence capable of hybridizing to a target HBB polynucleotide, (ii) a stem sequence;
  • a tracrRNA segment comprising a nucleotide sequence that is partially or completely complementary to the stem sequence
  • the guide sequence consists of 20-N nucleotides, where N is an integer between ⁇ 10 and 10, and the guide sequence comprises at least one specificity-enhancing modification at a nucleotide selected from positions 4-N, 5-N, 7-N, 9-N, 10-N, and 11-N, and wherein the synthetic guide RNA has gRNA functionality.
  • the synthetic guide RNA of any one of embodiments N1 to N7, wherein the at least one specificity-enhancing modification comprises 2′-O-methyl-3′-phosphonoacetate (MP), 2′-O-methyl-3′-thiophosphonoacetate (MSP), 2′-deoxy-3′-phosphonoacetate (DP), 2′-deoxy-3′-thiophosphonoacetate (DSP), or a combination thereof.
  • MP 2′-O-methyl-3′-phosphonoacetate
  • MSP 2′-O-methyl-3′-thiophosphonoacetate
  • DP 2′-deoxy-3′-phosphonoacetate
  • DSP 2′-deoxy-3′-thiophosphonoacetate
  • a method for cleaving, nicking or binding a target polynucleotide comprising:
  • RNA comprising:
  • a crRNA segment comprising (i) a guide sequence capable of hybridizing to a target polynucleotide, (ii) a stem sequence;
  • a tracrRNA segment comprising a nucleotide sequence that is partially or completely complementary to the stem sequence
  • the guide sequence consists of 20-N nucleotides, where N is an integer between ⁇ 10 and 10, and the guide sequence comprises at least one specificity-enhancing modification at a nucleotide selected from positions 4-N, 5-N, 7-N, 9-N, 10-N, and 11-N; and
  • gRNA:Cas protein complex comprising a Cas protein and the synthetic guide RNA
  • the target polynucleotide is selected from the group consisting of VEGFA polynucleotide, IL2RG polynucleotide, CLTA1 polynucleotide, and a CLTA4 polynucleotide.
  • a synthetic guide RNA comprising:
  • a crRNA segment comprising (i) a guide sequence capable of hybridizing to a target polynucleotide, (ii) a stem sequence;
  • a tracrRNA segment comprising a nucleotide sequence that is partially or completely complementary to the stem sequence
  • the guide sequence consists of 20-N nucleotides, where N is an integer between ⁇ 10 and 10, and the guide sequence comprises at least one specificity-enhancing modification at a nucleotide selected from positions 4-N, 5-N, 7-N, 9-N, 10-N, and 11-N, and wherein the synthetic guide RNA has gRNA functionality.
  • the synthetic guide RNA of any one of embodiments P1 to P8, wherein the specificity-enhancing modifications comprise 2′-O-methyl-3′-phosphonoacetate (MP), 2′-O-methyl-3′-thiophosphonoacetate (MSP), 2′-deoxy-3′-phosphonoacetate (DP), 2′-deoxy-3′-thiophosphonoacetate (DSP), or a combination thereof.
  • MP 2′-O-methyl-3′-phosphonoacetate
  • MSP 2′-O-methyl-3′-thiophosphonoacetate
  • DP 2′-deoxy-3′-phosphonoacetate
  • DSP 2′-deoxy-3′-thiophosphonoacetate
  • the synthetic guide RNA of embodiment P10, wherein the 2′-modification is selected from 2′-F and 2′-O-(2-methoxyethyl).
  • a method for cleaving, nicking or binding a target polynucleotide comprising:
  • RNA comprising:
  • a crRNA segment comprising (i) a guide sequence capable of hybridizing to a target polynucleotide, (ii) a stem sequence;
  • a tracrRNA segment comprising a nucleotide sequence that is partially or completely complementary to the stem sequence
  • the guide sequence comprises at least two consecutive specificity-enhancing modifications
  • gRNA:Cas protein complex comprising a Cas protein and the synthetic guide RNA
  • the at least one specificity-enhancing modification weakens hybridization between the guide sequence and the target polynucleotide.
  • a synthetic guide RNA comprising:
  • a crRNA segment comprising (i) a guide sequence capable of hybridizing to a target polynucleotide, (ii) a stem sequence;
  • a tracrRNA segment comprising a nucleotide sequence that is partially or completely complementary to the stem sequence
  • the guide sequence comprises at least two consecutive specificity-enhancing modifications, and wherein the synthetic guide RNA has gRNA functionality, and
  • the at least one specificity-enhancing modification weakens hybridization between the guide sequence and the target polynucleotide.
  • the synthetic guide RNA of any one of embodiments R1 to R10, wherein the specificity-enhancing modifications comprise 2′-O-methyl-3′-phosphonoacetate (MP), 2′-O-methyl-3′-thiophosphonoacetate (MSP), 2′-deoxy-3′-phosphonoacetate (DP), 2′-deoxy-3′-thiophosphonoacetate (DSP), or a combination thereof.
  • MP 2′-O-methyl-3′-phosphonoacetate
  • MSP 2′-O-methyl-3′-thiophosphonoacetate
  • DP 2′-deoxy-3′-phosphonoacetate
  • DSP 2′-deoxy-3′-thiophosphonoacetate
  • R16 The synthetic guide RNA of any one of embodiments R1 to R13, wherein the at least one specificity-enhancing modification strengthens hybridization between the guide sequence and the target polynucleotide and weakens hybridization between the guide sequence and an off-target polynucleotide.
  • R17 The synthetic guide RNA of any one of embodiments R1 to R16, wherein the guide RNA is a synthetic single guide RNA.
  • a method of selecting a synthetic guide RNA comprising:
  • first synthetic guide RNA and a second synthetic guide RNA each comprising:
  • a crRNA segment comprising (i) a guide sequence capable of hybridizing to a target polynucleotide, (ii) a stem sequence;
  • a tracrRNA segment comprising a nucleotide sequence that is partially or completely complementary to the stem sequence
  • each of the guide sequences consists of 20-N nucleotides, where N is an integer between ⁇ 10 and 10, counted from the 5′ end of the guide sequence,
  • first synthetic guide RNA comprises a specificity-enhancing modification at a first position within the guide sequence
  • second synthetic guide RNA comprises a specificity-enhancing modification at a second position within the guide sequence
  • a first gRNA:Cas protein complex comprising a Cas protein and the first synthetic guide RNA, contacting the target polynucleotide with the first gRNA:Cas protein complex, and cleaving, nicking or binding the target polynucleotide;
  • a second gRNA:Cas protein complex comprising a Cas protein and the second synthetic guide RNA, contacting the target polynucleotide with the second gRNA:Cas protein complex, and cleaving, nicking or binding the target polynucleotide;
  • any one of embodiments S1 to S7, wherein the method comprises providing a first through twentieth synthetic guide RNA comprising a specificity-enhancing modification at different nucleotide positions in the guide sequence portions, forming a gRNA:Cas protein complex using each of the synthetic guide RNAs, contacting the target polynucleotide with the gRNA:Cas protein complex, cleaving, nicking or binding the target polynucleotide and measuring the specificity of each synthetic guide RNA, and identifying one or more modified positions that provide the greatest specificity enhancement.
  • a kit for selecting a synthetic guide RNA comprising:
  • At least two synthetic guide RNAs comprising:
  • a crRNA segment comprising (i) a guide sequence capable of hybridizing to a target polynucleotide, (ii) a stem sequence;
  • a tracrRNA segment comprising a nucleotide sequence that is partially or completely complementary to the stem sequence
  • the guide sequence consists of 20-N nucleotides, where N is an integer between ⁇ 10 and 10, counted from the 5′ end of the guide sequence, and comprises at least one specificity-enhancing modification at a nucleotide in the guide sequence; and wherein the at least two synthetic guide RNAs differ from each other by having at least one different specificity-enhancing modification or by having the specificity-enhancing modification at least one different position in the guide sequence; and
  • Cas protein or a polynucleotide encoding said Cas protein.
  • kits of embodiment T2 wherein the kit comprises at least twenty synthetic guide RNAs.
  • kits of embodiment T1 or T2, wherein the Cas protein is Cas9 or Cpf1.
  • T4 The kit of any one of embodiments T1 to T3, wherein the specificity-enhancing modification comprises 2′-O-methyl-3′-phosphonoacetate (MP), 2′-O-methyl-3′-thiophosphonoacetate (MSP), 2′-deoxy-3′-phosphonoacetate (DP), or 2′-deoxy-3′-thiophosphonoacetate (DSP).
  • MP 2′-O-methyl-3′-phosphonoacetate
  • MSP 2′-O-methyl-3′-thiophosphonoacetate
  • DP 2′-deoxy-3′-phosphonoacetate
  • DSP 2′-deoxy-3′-thiophosphonoacetate
  • T5 The kit of any one of embodiments T1 to T3, wherein the specificity-enhancing modification comprises a 2′-modification that confers a C3′-endo sugar pucker and a phosphonoacetate or thiophosphonoacetate linkage modification.
  • T6 The kit of embodiment T5, wherein the 2′-modification is selected from 2′-F and 2′-O-(2-methoxyethyl).
  • T7 The kit of any one of embodiments T1 to T6, wherein the guide RNAs are synthetic single guide RNAs.
  • X embodiments means all the embodiments of which the numbers start with an X.
  • a synthetic guide RNA comprising:
  • a synthetic crRNA comprising (i) a guide sequence capable of hybridizing to a target polynucleotide, wherein the target polynucleotide comprises a target sequence adjacent to a PAM site, and (ii) a stem sequence;
  • the guide sequence consists of 20-N nucleotides, where N is an integer between ⁇ 10 and 10;
  • the guide sequence comprises at least one modification.
  • SP thiophosphonoacetate internucleotide linkage
  • the synthetic guide RNA or crRNA of embodiment X10, wherein said 2′-modification is selected from 2′-O-methyl, 2′-fluoro, and 2′-O-(2-methoxyethyl).
  • MP 2′-O-methyl-3′-phosphonoacetate
  • MSP 2′-O-methyl-3′-thiophosphonoacetate
  • X20 The synthetic guide RNA or crRNA of any one of embodiments X, X1 and X16-X19, wherein the at least one modification comprises a phosphonocarboxylate or thiophosphonocarboxylate internucleotide linkage modification at position 7-N of the guide sequence.
  • X22 The synthetic guide RNA or crRNA of any one of embodiments X, X1 and X16-X21, wherein the at least one modification comprises a phosphonocarboxylate or thiophosphonocarboxylate internucleotide linkage modification at position 9-N of the guide sequence.
  • X25 The synthetic guide RNA or crRNA of any one of embodiments X, X1 and X16-X24, wherein the at least one modification comprises a phosphonocarboxylate or thiophosphonocarboxylate internucleotide linkage modification at position 12-N of the guide sequence.
  • X27 The synthetic guide RNA or crRNA of any one of embodiments X, X1 and X16-X26, wherein the at least one modification comprises a phosphonocarboxylate or thiophosphonocarboxylate internucleotide linkage modification at position 14-N of the guide sequence.
  • X28 The synthetic guide RNA or crRNA of any one of embodiments X, X1 and X16-X27, wherein the at least one modification comprises a phosphonocarboxylate or thiophosphonocarboxylate internucleotide linkage modification at position 16-N of the guide sequence.
  • X29 The synthetic guide RNA or crRNA of any one of embodiments X, X1 and X16-X28, wherein the at least one modification comprises a phosphonocarboxylate or thiophosphonocarboxylate internucleotide linkage modification at position 17-N of the guide sequence.
  • X32 The synthetic guide RNA or crRNA of any one of embodiments X, X1 and X16-X31, wherein the at least one modification comprises a phosphonocarboxylate or thiophosphonocarboxylate internucleotide linkage modification at position 20-N of the guide sequence.
  • X34 The synthetic guide RNA or crRNA of any one of embodiments X, X1 and X16-X32, wherein the at least one modification comprises a thiophosphonocarboxylate internucleotide linkage.
  • X35 The synthetic guide RNA or crRNA of any one of embodiments X, X1 and X16-X33, wherein said phosphonocarboxylate internucleotide linkage is a phosphonoacetate linkage (P).
  • X36 The synthetic guide RNA or crRNA of any one of embodiments X, X1 and X16-X33, wherein said phosphonocarboxylate internucleotide linkage is a thiophosphonoacetate linkage (SP).
  • SP thiophosphonoacetate linkage
  • X40 The synthetic guide RNA or crRNA of any one of embodiments X-X38, wherein said target polynucleotide is located within an IL2RG polynucleotide.
  • the synthetic guide RNA or crRNA of embodiment X39, wherein the target polynucleotide comprises GCCCCACAGGGCAGTAA (SEQ ID NO:8).
  • the synthetic guide RNA or crRNA of embodiment X43, wherein the target polynucleotide comprises GATGTAGTGTTTCCACA (SEQ ID NO:4).
  • X60 The synthetic guide RNA or crRNA or method of any one of embodiments X-X58, wherein N equals 1, and the guide sequence consists of 19 nucleotides.
  • X65 The synthetic guide RNA or crRNA or method of any one of embodiments X-X58, wherein N equals ⁇ 1, and the guide sequence consists of 21 nucleotides.
  • X66 The synthetic guide RNA or crRNA or method of any one of embodiments X-X58, wherein N equals ⁇ 2, and the guide sequence consists of 22 nucleotides.
  • X68 The synthetic guide RNA or crRNA or method of any one of embodiments X-X58, wherein N equals ⁇ 4, and the guide sequence consists of 24 nucleotides.
  • RNA82 The synthetic guide RNA or crRNA or method of any of the preceding X embodiments, wherein the guide RNA has higher specificity for the target polynucleotide or higher gRNA functionality than a corresponding guide RNA without the modification.
  • the synthetic guide RNA or crRNA or method of any of the preceding X embodiments, wherein the at least one modification comprises 2′-deoxy-3′-phosphonoacetate (DP).
  • the synthetic guide RNA of embodiment Y10 wherein said guide RNA is a single guide RNA and wherein said at least one modification at the 5′-end, 3′-end or both ends of said guide RNA is independently selected from a 2′-O-methyl (M), a phosphorothioate internucleotide linkage (S), a phosphonoacetate internucleotide linkage (P), a thiophosphonoacetate internucleotide linkage (SP), a 2′-O-methyl-3′-phosphoroatioate (MS), a 2′-O-methyl-3′-phosphosphonoacetate (MP) and a 2′-O-methyl-3′-thiophosphosphonoacetate (MSP), or a combination thereof.
  • M 2′-O-methyl
  • S phosphorothioate internucleotide linkage
  • P phosphonoacetate internucleotide linkage
  • SP thiophosphonoacetate internucle
  • a synthetic guide RNA comprising:
  • a method for enhancing the specificity of a CRISPR function comprising:
  • Y29 The method of embodiment Y28, wherein the target polynucleotide comprises GCCCCACAGGGCAGTAA (SEQ ID NO:8) of the HBB gene, TAATGATGGCTTCAACA (SEQ ID NO:8) of the IL2RG gene, or GAGTGAGTGTGTGCGTG (SEQ ID NO:192) of the VEGFA gene.
  • the target polynucleotide comprises GCCCCACAGGGCAGTAA (SEQ ID NO:8) of the HBB gene, TAATGATGGCTTCAACA (SEQ ID NO:8) of the IL2RG gene, or GAGTGAGTGTGTGCGTG (SEQ ID NO:192) of the VEGFA gene.

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KR1020197000571A KR102382772B1 (ko) 2016-06-08 2017-06-08 화학적으로 변형된 가이드 rna를 사용하는 고 특이성 게놈 편집
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